DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

Auto‐phosphorylation of a voltage‐gated K+ channel controls non‐associative learning

Here, we characterize a new K⁺ channel–kinase complex that operates in the metazoan Caenorhabditis elegans to control learning behaviour. This channel is composed of a pore‐forming subunit, dubbed KHT‐1 (73% homology to human Kv3.1), and the accessory subunit MPS‐1, which shows kinase activity. Genetic, biochemical and electrophysiological evidence show that KHT‐1 and MPS‐1 form a complex in vitro and in native mechanosensory PLM neurons, and that KHT‐1 is a substrate for the kinase activity of MPS‐1. Behavioural analysis further shows that the kinase activity of MPS‐1 is specifically required for habituation to repetitive mechanical stimulation. Thus, worms bearing an inactive MPS‐1 variant (D178N) respond normally to touch on the body but do not habituate to repetitive mechanical stimulation such as tapping on the side of the Petri dish. Hence, the phosphorylation status of KHT‐1–MPS‐1 seems to be linked to distinct behavioural responses. In the non‐phosphorylated state the channel is necessary for the normal function of the touch neurons. In the auto‐phosphorylated state the channel acts to induce neuronal adaptation to mechanical stimulation. Taken together, these data establish a new mechanism of dynamic regulation of electrical signalling in the nervous system.     

Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.     

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA_ARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA_A autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca²⁺ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl⁻]_i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABA_A channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na_v-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA_A autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.     

Canalization and developmental instability of the fetal skull in a mouse model of maternal nutritional stress

Nutritional imbalance is one of the main sources of stress in both extant and extinct human populations. Restricted availability of nutrients is thought to disrupt the buffering mechanisms that contribute to developmental stability and canalization, resulting in increased levels of fluctuating asymmetry (FA) and phenotypic variance among individuals. However, the literature is contradictory in this regard. This study assesses the effect of prenatal nutritional stress on FA and among‐individual variance in cranial shape and size using a mouse model of maternal protein restriction. Two sets of landmark coordinates were digitized in three dimensions from skulls of control and protein restricted specimens at E17.5 and E18.5. We found that, by the end of gestation, maternal protein restriction resulted in a significant reduction of skull size. Fluctuating asymmetry in size and shape exceeded the amount of measurement error in all groups, but no significant differences in the magnitude of FA were found between treatments. Conversely, the pattern of shape asymmetry was affected by the environmental perturbation since the angles between the first eigenvectors extracted from the covariance matrix of shape asymmetric component of protein restricted and control groups were not significantly different from the expected for random vectors. In addition, among‐individual variance in cranial shape was significantly higher in the protein restricted than the control group at E18.5. Overall, the results obtained from a controlled experiment do not support the view of fluctuating asymmetry of cranial structures as a reliable index for inferring nutritional stress in human populations. Am J Phys Anthropol 154:544–553, 2014. © 2014 Wiley Periodicals, Inc.     

Ocular-Motor Profile and Effects of Memantine in a Familial Form of Adult Cerebellar Ataxia with Slow Saccades and Square Wave Saccadic Intrusions

Fixation instability due to saccadic intrusions is a feature of autosomal recessive spinocerebellar ataxias, and includes square wave intrusions (SWI) and macrosaccadic oscillations (MSO). A recent report suggested that the non-competitive antagonist of NMDA receptors, memantine, could decrease MSO and improve fixation in patients with spinocerebellar ataxia with saccadic intrusions (SCASI). We similarly tested two sisters, respectively of 58 and 60 years, with an unrecognized form of recessive, adult-onset cerebellar ataxia, peripheral neuropathy and slow saccades, who showed prominent SWI and also complained with difficulty in reading. We tested horizontal visually guided saccades (10°–18°) and three minutes of steady fixation in each patient and in thirty healthy controls. Both patients showed a significant reduction of peak and mean velocity compared with control subjects. Large SWI interrupting steady fixation were prominent during steady fixation and especially following visually guided saccades. Eye movements were recorded before and during the treatment with memantine, 20 mg/daily for 6 months. The treatment with memantine reduced both the magnitude and frequency of SWI (the former significantly), but did not modified neurological conditions or saccade parameters. Thus, our report suggests that memantine may have some general suppressive effect on saccadic intrusions, including both SWI and MSO, thereby restoring the capacity of reading and visual attention in these and in other recessive forms of ataxia, including Friedreich’s, in which saccadic intrusions are prominent.