Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Genome Wide Analysis of Fertility and Production Traits in Italian Holstein Cattle

A genome wide scan was performed on a total of 2093 Italian Holstein proven bulls genotyped with 50K single nucleotide polymorphisms (SNPs), with the objective of identifying loci associated with fertility related traits and to test their effects on milk production traits. The analysis was carried out using estimated breeding values for the aggregate fertility index and for each trait contributing to the index: angularity, calving interval, non-return rate at 56 days, days to first service, and 305 day first parity lactation. In addition, two production traits not included in the aggregate fertility index were analysed: fat yield and protein yield. Analyses were carried out using all SNPs treated separately, further the most significant marker on BTA14 associated to milk quality located in the DGAT1 region was treated as fixed effect. Genome wide association analysis identified 61 significant SNPs and 75 significant marker-trait associations. Eight additional SNP associations were detected when SNP located near DGAT1 was included as a fixed effect. As there were no obvious common SNPs between the traits analyzed independently in this study, a network analysis was carried out to identify unforeseen relationships that may link production and fertility traits.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

24-Epibrassinolide Mechanisms Regulating Blossom-End Rot Development in Tomato Fruit

Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca²⁺ content in the distal fruit tissue. However, many other factors can also determine fruit susceptibility to BER. It is possible that during fruit growth, Ca²⁺ imbalance can increase membrane leakiness, which may trigger the accumulation of reactive oxygen species, leading to cell death. Brassinosteroids are a class of plant hormones involved in stress defenses, specially increasing the activity of antioxidant enzymes and the accumulation of antioxidant compounds, such as ascorbic acid. The objective of this study was to understand the mechanisms by which 24-epibrassinolide (EBL) reduces fruit susceptibility to BER. Tomato plants ‘BRS Montese’ were cultivated in a greenhouse and were weekly sprayed with water (control) or EBL (0.01 µM) after full bloom. Plants and fruits were evaluated at 15 days after pollination (DAP). According to the results, EBL treatment inhibited BER development, increased fruit diameter, length, and fresh weight. EBL-treated fruit showed higher concentrations of soluble Ca²⁺ and lower concentrations of cell wall-bound Ca²⁺. EBL-treated fruit also had higher concentrations of ascorbic acid and lower concentrations of hydrogen peroxide, compared to water-treated fruit. EBL treatment increased the activity of the three main antioxidant enzymes known as ascorbate peroxidase, catalase, and superoxide dismutase. According to the results, EBL treatment maintained higher soluble Ca²⁺ and antioxidant capacity, reducing fruit susceptibility to BER.

     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Statistical independence and neural computation in the leech ganglion

 In this report, the input/output relations in an isolated ganglion of the leech Hirudo medicinalis were studied by simultaneously using six or eight suction pipettes and two intracellular electrodes. Sensory input was mimicked by eliciting action potentials in mechanosensory neurons with intracellular electrodes. The integrated neural output was measured by recording extracellular voltage signals with pipettes sucking the roots and the connectives. A single evoked action potential activated electrical activity in at least a dozen different neurons, some of which were identified. This electrical activity was characterized by a high degree of temporal and spatial variability. The action potentials of coactivated neurons, i.e. activated by the same mechanosensory neuron, did not show any significant pairwise correlation. Indeed, the analysis of evoked action potentials indicates clear statistical independence among coactivated neurons, presumably originating from the independence of synaptic transmission at distinct synapses. This statistical independence may be used to increase reliability when neuronal activity is averaged or pooled. It is suggested that statistical independence among coactivated neurons may be a usual property of distributed processing of neuronal networks and a basic feature of neural computation. Received: 20 September 1999 / Accepted in revised form: 3 March 2000     

Functional and Spectroscopic Characterization of Chlamydomonas reinhardtii Truncated Hemoglobins

The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO₂⁻ binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO₂⁻ concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO₂−binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Advances in molecular tools for the use of Zygosaccharomyces bailii as host for biotechnological productions and construction of the first auxotrophic mutant

The nonconventional yeast Zygosaccharomyces bailii has been proposed as a new host for biotechnological processes due to convenient properties such as its resistance to high sugar concentrations, relatively high temperatures and especially to acidic environments. We describe a series of new expression vectors specific for Z. bailii and the resulting improvements in production levels. By exploiting the sequences of the endogenous plasmid pSB2, 2microm-like multicopy vectors were obtained, giving a fivefold increase in production. A specific integrative vector was developed which led to 100% stability in the absence of selective pressure; a multiple-integration vector was constructed, based on an rRNA gene unit portion cloned and sequenced for this purpose, driving the insertion of up to 80 copies of the foreign construct. Moreover, we show the construction of the first stable auxotrophic mutant of Z. bailii, obtained by targeted gene deletion applied to ZbLEU2. The development of molecular tools for the Z. bailii manipulation has now reached a level that may be compatible with its industrial exploitation; the production of organic acids is a prominent field of application.