Investigating the multibudded and binucleate phenotype of the yeast Zygosaccharomyces bailii growing on minimal medium

The yeast Zygosaccharomyces bailii , known to have peculiar resistance to several environmental constraints, is very little known with respect to its genetics and life cycle. In addition to molecular and biochemical studies, cytofluorimetric and morphological analyses can also add information necessary to shed light on its interesting features. In the present study, the DNA and protein content as well as the cellular morphology of Z. bailii populations growing in minimal medium supplemented with different carbon sources and with the addition of different organic acids were investigated. The results show the occurrence of a multibudded phenotype and of a low, but significant percentage of binucleate cells occurring in the early-stationary phase. These traits appear to be different in comparison with the better-known laboratory yeast Saccharomyces cerevisiae . Experiments and speculations about these features and possible implications with Z. bailii main characteristics are discussed.     

Replication slippage versus point mutation rates in short tandem repeats of the human genome

Short tandem repeats (STRs) are subjected to two kinds of mutational modifications: point mutations and replication slippages. The latter is found to be the more frequent cause of STR modifications, but a satisfactory quantitative measure of the ratio of the two processes has yet to be determined. The comparison of entire genome sequences of closely enough related species enables one to obtain sufficient statistics by counting the differences in the STR regions. We analyzed human–chimpanzee DNA sequence alignments to obtain the counts of point mutations and replication slippage modifications. The results were compared with the results of a computer simulation, and the parameters quantifying the replication slippage probability as well as the probabilities of point mutations within the repeats were determined. It was found that within the STRs with repeated units consisting of one, two or three nucleotides, point mutations occur approximately twice as frequently as one would expect on the basis of the 1.2% difference between the human and chimpanzee genomes. As expected, the replication slippage probability is negligible below a 10-bp threshold and grows above this level. The replication slippage events outnumber the point mutations by one or two orders of magnitude, but are still lower by one order of magnitude relative to the mutability of the markers that are used for genotyping purposes.     

Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.     

Photosynthesis and carbon metabolism in plantain (Musa AAB) plantlets growing in temporary immersion bioreactors and during ex vitro acclimatization

Summary The photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO₂m⁻²s⁻¹); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.     

Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis

Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1–q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor β (OSMRβ), in three families. OSMRβ is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.     

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus

The role played by Ca²⁺ ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca²⁺ ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca²⁺ ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca²⁺ ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of β-sheet rich hIAPP aggregates.     

Uncoupling of protein-3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells.

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6 cell line. UCP3 was expressed in relatively low levels (< 1 microg x mg(-1) membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X-100 extraction under conditions that fully solubilize membrane bound proteins. Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle-derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.     

Bacterial Community Structure and Location in Stilton Cheese

The microbial diversity occurring in Stilton cheese was evaluated by 16S ribosomal DNA analysis with PCR-denaturing gradient gel electrophoresis. DNA templates for PCR experiments were directly extracted from the cheese as well as bulk cells harvested from a variety of viable-count media. The variable V3 and V4-V5 regions of the 16S genes were analyzed. Closest relatives of Lactococcus lactis, Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus curvatus, Leuconostoc mesenteroides, Staphylococcus equorum, and Staphylococcus sp. were identified by sequencing of the DGGE fragments. Fluorescently labeled oligonucleotide probes were developed to detect Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides in fluorescence in situ hybridization (FISH) experiments, and their specificity for the species occurring in the community of Stilton cheese was checked in FISH experiments carried out with reference cultures. The combined use of these probes and the bacterial probe Eub338 in FISH experiments on Stilton cheese sections allowed the assessment of the spatial distribution of the different microbial species in the dairy matrix. Microbial colonies of bacteria showed a differential location in the different parts of the cheese examined: the core, the veins, and the crust. Lactococci were found in the internal part of the veins as mixed colonies and as single colonies within the core. Lactobacillus plantarum was detected only underneath the surface, while Leuconostoc microcolonies were homogeneously distributed in all parts observed. The combined molecular approach is shown to be useful to simultaneously describe the structure and location of the bacterial flora in cheese. The differential distribution of species found suggests specific ecological reasons for the establishment of sites of actual microbial growth in the cheese, with implications of significance in understanding the ecology of food systems and with the aim of achieving optimization of the fermentation technologies as well as preservation of traditional products.     

Field performance of artificial seed-derived sugarcane plants

This study shows the behaviour of sugarcane plants cv. CP-5243 derived from artificial seed compared with traditional and isolated bud methods. Artificial seed-acclimatised plants were planted in field conditions simultaneously with two-control treatments previously germinated: macropropagated plants derived from stems of three buds and axillary buds isolated from field-grown plants. Plants from artificial seed were taller and had a smaller diameter at 8 months, but these differences disappeared at 12 months. With respect to sugar analysis and yield, no differences in all parameters evaluated were found between artificial seed-derived plants and plants derived from the two other methods.     

Harder,better, faster,stronger: Weapon size is more sexually dimorphic than weapon biomechanical components in two freshwater anomuran species

Sexual selection influences the evolution of morphological traits that increase the likelihood of monopolizing scarce resources. When such traits are used during contests, they are termed weapons. Given that resources are typically linked to monopolizing mating partners, theory expects only males to bear weapons. In some species, however, females also bear weapons, although typically smaller than male weapons. Understanding why females bear smaller weapons can thus help us understand the selective pressures behind weapon evolution. However, most of our knowledge comes from studies on weapon size, while the biomechanics of weapons, such as the size of the muscles, efficiency, and shape are seldom studied. Our goal was to test if the theoretical expectations for weapon size sexual dimorphism also occur for weapon biomechanics using two aeglid crab species. Males of both species had larger claws which were also stronger than female claws. Male claws were also more efficient than females' claws (although we used only one species in this analysis). For weapon shape, though, only one species differed in the mean claw shape. Regarding scaling differences, in both species, male claws had higher size scaling than females, while only one species had a higher shape scaling. However, male weapons did not have higher scaling regarding strength and efficiency than females. Thus, males apparently allocate more resources in weapons than females, but once allocated, muscle and efficiency follow a similar developmental pathway in both sexes. Taken together, our results show that sexual dimorphism in weapons involves more than differences in size. Shape differences are especially intriguing because we cannot fully understand its causes. Yet, we highlight that such subtle differences can only be detected by measuring and analysing weapon shape and biomechanical components. Only then we might better understand how weapons are forged.