Cdc7 is required throughout the yeast S phase to activate replication origins

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7^ts allele were first arrested in G₁ at the cdc7 block by incubation at 37°C, and then were allowed to enter S phase by brief incubation at 23°C. During the S phase, after return to 37°C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.     

Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis

Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Evaluation of a Standard Protocol for Retentive Encopresis: A Replication

Replicated the efficacy of a short-term, combined medical andbehavioral intervention protocol for retentive encopresis. Fifty-ninechildren who had failed standard medical management for retentiveencopresis and their parents participated in six 1-hour grouptreatment sessions. Treatment protocol combined the medicalmanagement strategies of enema clean out, increasing dietaryfiber, and daily toilet sitting with the child behavior managementstrategies of differential attention, contingency management,and contracting. For the overall sample, the number of soilingincidents decreased 85%, the weekly frequency of independentbowel movements increased 15%, the weekly frequency of parent-promptedbowel movements increased 9%, and daily dietary fiber intakeincreased 121% pre-to posttreatment. The majority of the sample(86%) stopped soiling by the end of treatment and did not requirefurther treatment. Results are discussed in terms of the comparabilitywith previous findings and the utility of combined medical andpsychological treatments for children with encopresis who havefailed standard medical approaches     

Excitation of units in the lateral geniculate and contiguous nuclei of the cat by stretch of extrinsic ocular muscles

Summary In cats, anaesthetized with chloralose and paralysed, the responses of units in the right lateral thalamus were recorded while the extrinsic ocular muscles (EOM) of the right eye were stretched in the dark. Phasic responses were found in all layers of the dorsal lateral geniculate nucleus (LGNd) and in the perigeniculate nucleus (PGN). A given unit usually responded to stretch of more than one EOM and thus to more than one direction of rotation of the eye in the orbit. LGNd. Of a sample of 76 units in LGNd, 55 (72%) gave visual but no muscle responses and 21 (28%) responded to EOM stretch. In all, 40 units with EOM responses were examined and 25 of the 27 tested (93%) also had visual responses. Of the 40 units, 32 could be allocated to layers, thus: layer A, 8 (25%); layer A1, 20 (63%); layer B, 3 (9%); central interlaminar nucleus, 1 (3%). It is interesting that most of the EOM responses were found in layer A1 which receives the excitatory visual input from the eye whose EOM were stretched. Muscle responsive units occurred with ON- and OFF-centre visual responses of sustained and transient types. PGN. In PGN, 21 units gave EOM responses and most of them were also excited by visual input.The conclusion is that the LGNd and PGN recieve an extraretinal proprioceptive signal which should be present during at least large saccadic eye movements. The anatomical pathways which may be involved and the significance of the signal are discussed briefly.     

The pathogenicity of long versus short fibre samples of amosite asbestos administered to rats by inhalation and intraperitoneal injection.

For many years it has been accepted that fibre dimensions are the most important factor in the development of asbestos related disease with long fibres being more dangerous than short for all types of asbestos. This information has been derived from in vitro experiments and injection or implantation experiments since the kilogramme quantities of specially prepared dusts that are necessary for long term inhalation have not been available. The present study has taken advantage of the availability of a sample of amosite produced so that almost all fibres were less than 5 micron in length. The effects of this dust were compared to dust prepared from raw amosite that contained a very high proportion of long fibres. Previous data from studies with UICC amosite, which was intermediate in length, were also available for comparison. At the end of 12 months of dust inhalation, significantly more short fibre amosite was present in the lung tissue compared to the long but while the long fibre dust caused the development of widespread pulmonary fibrosis, no fibrosis at all was found in animals treated with short fibre. One third of animals treated with long fibre dust developed pulmonary tumours or mesotheliomas but no pulmonary neoplasms were found in animals treated with short fibre dust. Following intraperitoneal injection, the long fibre amosite produced mesotheliomas in 95% of animals with a mean induction period of approximately 500 days. With short fibre dust, only a single mesothelioma developed after 837 days. In previous inhalation studies with UICC amosite, relatively little pulmonary fibrosis had developed and only two benign pulmonary tumours. This would suggest that to produce a significant carcinogenic response in rat lung tissue amosite fibres must be longer than those in the UICC preparation. Following the injection of UICC amosite, however, mesotheliomas developed in the same proportion of animals and with the same mean induction period as with long fibre dust. From this it would appear that while very short fibres exhibit little carcinogenicity to either lung or mesothelial tissues, mesotheliomas can be produced by dust preparations consisting of shorter fibres than are needed to produce tumours.     

Compartmentalized immune responses: antigen-specificity of cerebrospinal fluid T-cell lines maintained in the absence of antigen

The generation of long-term interleukin 2-dependent T-cell lines from anatomically compartmentalized sites of pathology offers a unique approach to the investigation of certain autoimmune diseases. However, it is generally believed that antigen-specific T-cell lines and clones lose antigen reactivity and specificity when propagated in the absence of antigen. Therefore, the optimal application of this approach to such diseases in which the pathogenetic antigens are unknown may be difficult. In approaching this problem, we have recently demonstrated that a proportion of antigen-specific T-cell lines derived from the peripheral circulation can maintain antigen specificity if propagated with antigen-presenting cells alone or with antigen-presenting cells together with OKT3 antibody, but in either case in the absence of antigen. In this report we describe the use of this approach to maintain the antigen specificity of T cells obtained from an anatomically compartmentalized site of pathology--the cerebrospinal fluid from a patient with tuberculous meningitis. We report here that a proportion of the T-cell lines generated from such cerebrospinal fluid lymphocytes can be maintained as antigen specific in the absence of antigen if propagated with either antigen-presenting cells alone or with antigen-presenting cells and OKT3 antibody. The approach illustrated in this report should now find broad applicability in the investigation of a number of autoimmune disease.