Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

Estrogen and progesterone receptor content in the pituitary gland and uterus of progesterone-primed and gonadotropin releasing hormone-treated anestrous ewes

The objective of this work was to investigate the effect of progesterone (P) and gonadotropin-releasing hormone (GnRH) treatment on estrogen receptor (ER) and P receptor (PR) concentrations in the pituitary gland and uterus of anestrous ewes. Ewes were either not treated (group C, n = 4); were treated with 0.33 g P-controlled internal drug release (P-CIDR) for 10 days (group P, n = 4), with GnRH, 6.7 ng i.v. injections every 2 h for 18 h followed by a 4 microg bolus administration of Receptal at 20 h (group GnRH, n = 4), or with a combination of the P and GnRH treatment (group P + GnRH, n = 3). Ewes were humanely killed either at the beginning of the experiment (group C), when the CIDR was removed (group P), or 24 h after the GnRH bolus treatment (groups GnRH and P + GnRH). Progesterone treatment increased serum P concentrations, indicating that the treatment was effective. All GnRH treated ewes had similar luteinizing hormone (LH) surges, which lasted 8 h. At slaughter, estradiol (E2) concentrations in the GnRH group were higher than in groups C, P, and P + GnRH. Treatment with GnRH increased more than 10-fold the content of ER and PR in the pituitary gland without altering steroid receptor concentrations in the uterus. When GnRH was combined with P the uterine receptor contents were higher than with P treatment alone. The treatment with P decreased ER and PR content in the uterus, but had no effect on the pituitary gland. The results show that regulation by P and GnRH of ER and PR content in anestrous ewes is tissue-specific.     

MICA-A5.1 allele is associated with atypical forms of celiac disease in HLA-DQ2-negative patients

We selected 38 consecutive celiac disease (CD) patients (from a group of 316 consecutive CD patients) and 91 healthy blood donors, all of whom were HLA-DQ2 (DQA1*0501/DQB1*0201) negative, and investigated the presence of the classically associated alleles HLA-DQ8 and HLA-DRB4. We also studied the distribution of MICA transmembrane alleles in the two clinical forms of the disease. For this reason, these 38 DQ2-negative patients were subdivided into two groups: 18 typical CD patients and 20 atypical CD patients. No differences were found in the distribution of the DRB4 allele between DQ2-negative patients and controls. The HLA-DQ8 heterodimer (DQA1*03xx/DQB1*0302) was increased in CD patients (29%) compared with controls (10%), but no statistical differences were found. No differences were observed in the frequency of these alleles between either group of CD DQ2-negative patients. MICA-A5.1 was increased in atypical CD patients when compared with the typical forms of disease ( P(c)=0.03) and with healthy controls (P(c)=0.002). No other MICA allele was found to be significantly increased in the groups under study. The presence of MICA-A5.1 in atypical CD DQ2-negative patients may indicate a possible role of this allele in the development of CD.     

Distribution of human virus contamination in shellfish from different growing areas in Greece,Spain, Sweden,and the United Kingdom

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.     

Malnutrition and its association with intestinal parasitism among children from a village in the Colombian Amazonian region

Intestinal parasites and malnutrition cause high morbidity among children in developing countries. A study to establish the associations between intestinal parasitism and undernourishment was undertaken in a village in the Colombian Amazon region. Two hundred thirty seven children each provided four stool samples for parasitic analysis. Anthropometric indices were calculated based on height and weight (HAZ), weight and age (WAZ) and height and weight (WHZ) using the international growth standards recommended by the World Health Organization. Of the 237 children, 86.1% were carrying parasites, 54% had 2 or more parasites, 30% were stunted, 10% were underweight, and 2.5% were wasting. HAZ and WAZ indices were inversely related to age and number of parasites. Socioeconomic status was positively correlated with the number of parasites and inversely related to the HAZ index (p < 0.05). In conclusion, frequencies of undernourishment and intestinal parasites among the sample population were higher than the national average for Colombia. As previous studies have indicated, age, intestinal parasitism and socio-economic level are variables significantly related to nutritional state.     

Stabilization of trypsin by chemical modification with β-cyclodextrin monoaldehyde

The monoaldehyde derivative of -cyclodextrin was attached to trypsin via reductive alkylation with NaBH₄. The thermostability was enhanced from 49.5 °C to 60 °C for modified trypsin. The activation free energy of thermal inactivation at 50 °C was increased by 3.2 kJ mol^–1. The conjugated enzyme retained 100% of its initial activity after 3 h incubation at pH 9.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.     

Cutting edge: dynamic redistribution of tetraspanin CD81 at the central zone of the immune synapse in both T lymphocytes and APC

The tetraspanin CD81 has been involved in T-dependent B cell-mediated immune responses. However, the behavior of CD81 during immune synapse (IS) formation has not been elucidated. We determined herein that CD81 redistributed to the contact area of T cell-B cell and T cell-dendritic cell conjugates in an Ag-dependent manner. Confocal microscopy showed that CD81 colocalized with CD3 at the central supramolecular activation complex. Videomicroscopy studies with APC or T cells transiently expressing CD81-green fluorescent protein (GFP) revealed that in both cells CD81 redistributed toward the central supramolecular activation complex. In T lymphocytes, CD81-GFP rapidly redistributed to the IS, whereas, in the APC, CD81-GFP formed a large accumulation in the contact area that later concentrated in a discrete cluster and waves of CD81 accumulated at the IS periphery. These results suggest a relevant role for CD81 in the topography of the IS that would explain its functional implication in T cell-B cell collaboration.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.     

Development and evolution of inner ear sensory epithelia and their innervation

The development and evolution of the inner ear sensory patches and their innervation is reviewed. Recent molecular developmental data suggest that development of these sensory patches is a developmental recapitulation of the evolutionary history. These data suggest that the ear generates multiple, functionally diverse sensory epithelia by dividing a single sensory primordium. Those epithelia will establish distinct identities through the overlapping expression of genes of which only a few are currently known. One of these distinctions is the unique pattern of hair cell polarity. A hypothesis is presented on how the hair cell polarity may relate to the progressive segregation of the six sensory epithelia. Besides being markers for sensory epithelia development, neurotrophins are also expressed in delaminating cells that migrate toward the developing vestibular and cochlear ganglia. These delaminating cells originate from multiple sites at or near the developing sensory epithelia and some also express neuronal markers such as NeuroD. The differential origin of precursors raises the possibility that some sensory neurons acquire positional information before they delaminate the ear. Such an identity of these delaminating sensory neurons may be used both to navigate their dendrites to the area they delaminated from, as well as to help them navigate to their central target. The navigational properties of sensory neurons as well as the acquisition of discrete sensory patch phenotypes implies a much more sophisticated subdivision of the developing otocyst than the few available gene expression studies suggest.