A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Non-Invasive monitoring of diaphragmatic timing by means of surface contact sensors: An experimental study in dogs

Background

The predictive role of many cytokines has not been well defined in Acute Respiratory Distress Syndrome (ARDS).

Methods

We measured prospectively IL-4, IL-6, IL-6 receptor, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid (BALF) in 59 patients who were admitted to ICU in order to identify predictive factors for the course and outcome of ARDS. The patients were divided into three groups: those fulfilling the criteria for ARDS (n = 20, group A), those at risk for ARDS and developed ARDS within 48 hours (n = 12, group B), and those at risk for ARDS but never developed ARDS (n = 27, group C).

Results

An excellent negative predictive value for ARDS development was found for IL-6 in BALF and serum (100% and 95%, respectively). IL-8 in BALF and IL-8 and IL-10 serum levels were higher in non-survivors in all studied groups, and were associated with a high negative predictive value. A significant correlation was found between IL-8 and APACHE score (r = 0.60, p < 0.0001). Similarly, IL-6 and IL-6r were highly correlated with PaO2/FiO2 (r = -0.27, p < 0.05 and r = -0.55, p < 0.0001, respectively).

Conclusions

BALF and serum levels of the studied cytokines on admission may provide valuable information for ARDS development in patients at risk, and outcome in patients either in ARDS or in at risk for ARDS.     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.     

The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity

The SET protein and the cell cycle inhibitor p21(Cip1) interact in vivo and in vitro. We identified here the domain (157)LIF(159) of p21(Cip1) as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21(Cip1), one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21(Cip1) co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G(2)/M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21(Cip1) cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G(2)/M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G(2)/M transition by modulating cyclin B-CDK1 activity.     

Do capuchin monkeys, Cebus apella, know what conspecifics do and do not see?

Capuchin monkeys were tested in five experiments in which two individuals competed over food. When given a choice between retrieving a piece of food that was visible or hidden from the dominant, subordinate animals preferred to retrieve hidden food. This preference is consistent with the hypotheses that either (1) the subordinate knew what the dominant could and could not see or (2) the subordinate was monitoring the behaviour of the dominant and avoiding the piece of food that it approached. To test between these alternatives, we released subordinates with a slight head start forcing them to make their choice (between a piece of food hidden or visible to the dominant) before the dominant entered the area. Unlike chimpanzees, Pan troglodytes, subordinates that were given a head start did not preferentially approach hidden pieces of food first. Therefore, our experiments provide little support for the hypothesis that capuchin monkeys are sensitive to what another individual does or does not see. We compare our results with those obtained with chimpanzees in the same paradigm and discuss the evolution of primate social cognition. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.      

Glucose 6-phosphate produced by gluconeogenesis and by glucokinase is equally effective in activating hepatic glycogen synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.     

Epidemiological study of cryptococcosis in Spain: first results

The study constitutes an approach to the knowledge of the epidemiology of cryptococosis in Spain. For detection of cases 167 Spanish hospitals were contacted. All cases included were accompanied by the correspondent isolate of Cryptococcus neoformans, together with clinical, demographic and mycological data. Results obtained from January 1998 to end of December 1999 are analysed and presented here. Fifty-six Spanish hospitals reported 58 cases of cryptococcosis; only 43 of them were adequately documented and accompanied by the clinical isolate. The results showed a higher incidence in males (88.4%) than in females (11.6%); being most frequently affected those between 30 and 40 years old (48.8%). The 84.6% (33) corresponded to new cases and 15.4% (6) to relapses of the disease. The HIV infection was the most frequent risk factor reported (86%) and, for 29.7% (11) of them, cryptococcosis was the AIDS defining disease. For the diagnosis, CSF analysis showed the best results (India ink; culture and antigen detection). All strains collected (100%) corresponded to C. neoformans variety neoformans. Serotypes distribution was 45.5% for serotype A and 22.7% for each of serotypes D and AD.     

Regulation of c-Ret,GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6-hydroxydopamine (6-OHDA) transiently increased c-Ret and GFRalpha1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c-Ret and GFRalpha1 was downregulated. GFRalpha2 expression was differentially regulated, as it decreased only 6 days after 6-OHDA injection. Triple-labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6-OHDA lesion.     

Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA

Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.     

Zonal distribution of glycogen synthesis in isolated rat hepatocytes

Incubation of hepatocytes isolated from fasted rats with [14C]glucose for short periods of time showed that the initial stages of glycogen synthesis occur near the plasma membrane. Incubation with [14C]glucose followed by cold glucose demonstrated that glycogen synthesis is always active at the hepatocyte periphery and that previously synthesised glycogen moves towards the centre of the cell, while its place is filled by newly synthesised molecules. However, the reverse experiment, incubation with cold glucose before addition of [14C]glucose, showed that, as glycogen synthesis progresses, it also becomes gradually active in more internal sites of the hepatocyte. These results indicate a spatial order in the synthesis of hepatic glycogen.