Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis

Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.     

The role of single-stranded DNA and polymerase alpha in establishing the ATR,Hus1 DNA replication checkpoint

Using a nucleus-free DNA replication system we have investigated the roles of Xenopus ATR (XATR) and Hus1 (Xhus1) as the DNA replication checkpoint sensors. Like XATR, Xhus1 is required for the checkpoint-dependent phosphorylation of Xchk1 and associates with chromatin in an initiation-dependent manner. While removal of replication protein A inhibits chromatin association of both XATR and Xhus1, removal of polymerase alpha only disrupts chromatin association of Xhus1. In addition, chromatin association of XATR and Xhus1 are independent of each other. Finally, like XATR, Xhus1 associates with chromatin in unperturbed S phase and dissociates from chromatin following completion of DNA replication.     

A role for Ran-GTP and Crm1 in blocking re-replication

All eukaryotic cells have regulatory mechanisms that limit genomic replication to a single round each cell cycle. These systems function by blocking formation of prereplication complexes. The regulatory mechanisms in the yeast S. cerevisiae have been identified, but these do not appear to be conserved in metazoans. Using Xenopus egg extracts, we have identified a metazoan-specific regulatory system that limits replication to a single round. We show that during S phase, soluble MCM helicase, an essential initiation factor, is inactivated when it associates with exportin-1/Crm1. Formation of this complex is dependent on both high Ran-GTP and cdk2 kinase activity. Lowering Ran-GTP within nuclei or nuclear extracts allows MCM to reassociate with chromatin during S phase and induces re-replication. Importantly, prevention of re-replication requires MCM-Crm1 complex formation, but it does not require export of MCM from the nucleus. Therefore, in metazoans, Crm1 functions in both nuclear export and blocking of re-replication.     

CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least approximately 2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.     

Inherited predisposition to mycobacterial infection: historical considerations

Although one third of the world's population is estimated to be infected with Mycobacterium tuberculosis, only one tenth of infected individuals develop clinical disease. There is substantial epidemiological evidence that host genetic factors are important determinants of susceptibility to mycobacterial disease. This paper gives a historical context to the recent exciting advances in the field which have led to the identification of a number of human mycobacterial susceptibility genes.     

Quantitative analysis of free sphingoid bases in the brain and spinal cord tissues by high-performance liquid chromatography with a fluorescence detection

The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography–fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform–methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 μl), 90–95% in 45 ng/tube of sphingosine and sphinganine within 2–12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.     

Same/Different Abstract Concept Learning by Archerfish (Toxotes chatareus)

While several phylogenetically diverse species have proved capable of learning abstract concepts, previous attempts to teach fish have been unsuccessful. In this report, the ability of archerfish (Toxotes chatareus) to learn the concepts of sameness and difference using a simultaneous two-item discrimination task was tested. Six archerfish were trained to either select a pair of same or different stimuli which were presented simultaneously. Training consisted of a 2-phase approach. Training phase 1: the symbols in the same and different pair did not change, thereby allowing the fish to solve the test through direct association. The fish were trained consecutively with four different sets of stimuli to familiarize them with the general procedure before moving on to the next training phase. Training phase 2: six different symbols were used to form the same or different pairs. After acquisition, same/different concept learning was tested by presenting fish with six novel stimuli (transfer test). Five fish successfully completed the first training phase. Only one individual passed the second training phase, however, transfer performance was consistent with chance. This individual was given further training using 60 training exemplars but the individual was unable to reach the training criterion. We hypothesize that archerfish are able to solve a limited version of the same/different test by learning the response to each possible stimulus configuration or by developing a series of relatively simple choice contingencies. We conclude that the simultaneous two-item discrimination task we describe cannot be successfully used to test the concepts of same and different in archerfish. In addition, despite considerable effort training archerfish using several tests and training methods, there is still no evidence that fish can learn an abstract concept-based test.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.