Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity.

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.

Reversible phosphorylation at the C-terminal regulatory domain of p21(Waf1/Cip1) modulates proliferating cell nuclear antigen binding.

The p53-inducible gene product p21(WAF1/CIP1) plays a critical role in regulating the rate of tumor incidence, and identifying mechanisms of its post-translational regulation will define key pathways that link growth control to p21-dependent tumor suppression. A eukaryotic cell model system has been developed to determine whether protein kinase signaling pathways that phosphorylate human p21 exist in vivo and whether such pathways regulate the binding of p21 to one of its key target proteins, proliferating cell nuclear antigen (PCNA). Although human p21 expressed in Sf9 cells is able to form a complex with human PCNA, the inclusion of cell-permeable phosphatase inhibitors renders p21 protein inactive for PCNA binding. The treatment of this inactive isoform of p21 with alkaline phosphatase restores its binding to PCNA, suggesting that p21 expressed in Sf9 cells is subject to reversible phosphorylation at a key regulatory site(s). A biochemical approach was subsequently used to map the phosphorylation sites within p21, whose modification in vitro can inhibit p21-PCNA complex formation, to the C-terminal domain at residues Thr(145) or Ser(146). A phospho-specific antibody was developed that only bound to full-length p21 protein after phosphorylation in vitro at Ser(146), and this reagent was further used to demonstrate that the inactive isoform of p21 recovered from Sf9 cells treated with phosphatase inhibitors had been phosphorylated in vivo at Ser(146). These data identify the first phosphorylation site within the C-terminal regulatory domain of p21 whose modification in vivo modulates p21-PCNA interactions and define a eukaryotic cell model that can be used to study post-translational signaling pathways that regulate p21.

Survival estimates for adults with cystic fibrosis born in the United Kingdom between 1947 and 1967. The UK Cystic Fibrosis Survey Management Committee.

BACKGROUND: The UK has published observed cohort survival figures for subjects with cystic fibrosis born since 1968. Prior to 1968 cohorts cannot be established directly from routine data as cystic fibrosis was classified with a number of unrelated conditions in ICD7. Reported here are interrupted survival curves from 1978 for patients with cystic fibrosis born before 1968. METHODS: Life tables for the three year cohorts born between 1947 and 1967 were constructed by firstly estimating the numbers of patients with cystic fibrosis born in each cohort from live birth data and the disease incidence. The number of the estimated cohort that had survived to 1978 is known, which enables the proportion surviving to 1978 to be calculated. The survival of these cohorts after 1978 can be calculated in the usual way. RESULTS: The survival for each successive cohort was better than that of the previous one, but most of the improvements appear to have taken place up to the age of about 20 years. Only 3% of the 1947-49 cohort survived to 30 years of age compared with 21% for the 1965-67 cohort, and 3% of the 1953-55 cohort survived to 40 years of age. For the later cohorts the mortality rate for those aged between 26 and 30 years appears to be about 50 per 1000 per year. CONCLUSIONS: While the trend in the numbers surviving into later adulthood is upwards, the mortality rates for these ages does not appear to be improving. It is not possible to tell from these data whether the high mortality rates in adulthood will improve with better resourced adult clinics or with improved treatment during childhood.

Incidence, population, and survival of cystic fibrosis in the UK, 1968-95. UK Cystic Fibrosis Survey Management Committee.

The UK Cystic Fibrosis Survey holds data on all people resident in the UK who were diagnosed as having cystic fibrosis and born either since 1968 or before 1968 and alive in 1977. Thus, incidence may be reported from 1968 and prevalence from 1977. The previous estimates are updated to the end of 1995 from data held in the database on 23 August 1996. The incidence is now calculated as one in 2415 live births. The 1992 mid-year population was 6500 people with 65% aged under 16 years. Births outnumber deaths by 160 per year, which suggests a population of 7750 by the year 2000, with all the increase being in the adult age range. The survival of successive cohorts continues to be better than earlier cohorts, the linear descent of the curves is still evident. The infant mortality rate for cystic fibrosis is now under 20 per thousand per year and early childhood mortality is under five per thousand per year. The crude mortality rate for 1995 was 21 per thousand per year, but the standardised mortality ratio was about 3300.

Height and weight in cystic fibrosis: a cross sectional study. UK Cystic Fibrosis Survey Management Committee.

Cross sectional data reporting the height, weight, and body mass index of UK patients with cystic fibrosis are presented. During the first decade of life height and weight in patients with cystic fibrosis are maintained at about 0.5 SD below those of the general population, which reflects an improvement over earlier published observations. Postpubertal stature and weight maintenance in the cystic fibrosis population still show substantial deficits which may be related to treatment.

Application of microcolumn liquid chromatography-continuous-flow fast atom bombardment mass spectrometry in environmental studies of sulfonylurea herbicides.

The use of 0.25-mm I.D. packed capillary liquid chromatography columns coupled with continuous-flow fast atom bombardment (FAB) mass spectrometry has proven to be a very valuable technique, especially for the identification of unknown sulfonylurea herbicide metabolites. Several new and unusual heterocycle ring-opened metabolites and hydrolysis products were identified, and metabolic pathways were proposed. Typical column flow-rates are 1-2 microliters/min, which allows direct coupling with no sample splitting. This is important in our metabolite identification work, since we are usually sample-limited. Techniques for increasing injection volume to allow analyses of dilute solutions and the use of polymeric packing for separation of polar metabolites are discussed. The FAB mass spectra usually provide unequivocal molecular weights and structurally useful fragments ions, which often allows structure assignments on exceedingly small quantities of isolated metabolites.

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. II. Partial biochemical characterization.

Culture supernatants of splenic T cells from susceptible CBA mice chronically infected with Trypanosoma cruzi contain a suppressive substance which can inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens. The suppressive substance is distinct from T. cruzi antigen inasmuch as the supernatant depleted of any residual T. cruzi antigen by an affinity column still retains the suppressive activity, whereas addition of T. cruzi antigens to control supernatant did not confer suppressive function. The suppressive supernatant does not contain detectable levels of IL-1, IL-2, IL-3, or IFN-gamma but a modest level of IL-1 and IL-2 inhibitory activities. However, both these inhibitory activities elute at a different position from the DTH suppressive activity on gel filtration. The DTH suppressive activity is heat labile (1 h, 56 degrees C), cryostable, but destroyed by trypsin treatment. It binds to ricin but not to lentil lectin. Sepharose 4B gel filtration and HPLC analysis in mild chaotropic agents (urea, ethylene glycol) demonstrate that the suppressive substance has an apparent Mr of 30 to 60 kDa, but full DTH-suppressive activity is retained only in an aggregated form.

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. I. Preferential inhibition of the induction of delayed-type hypersensitivity.

Culture supernatants of spleen cells from susceptible CBA mice chronically infected with Trypanosoma cruzi were able to inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens as measured by 24-hr footpad swelling, bone marrow homing, and radioactivity accumulation assays. The suppressive activity, which was also present in the serum of these chronically infected mice, appears to be specific for the induction of DTH and had no effect on the 3-hr immediate-type hypersensitivity. It also failed to modify the expression of DTH in presensitized mice. Furthermore, it did not affect the synthesis in normal recipients of specific antibody or the induction of helper T cells or cytotoxic T cells. It also failed to induce DTH tolerance as recipient mice with markedly reduced DTH were able to develop a normal DTH response after secondary immunization. The suppressive activity was produced by an Ig- macrophage-depleted splenic T cell population, whose capacity to secrete the suppressive substance was completely abrogated by treatment in vitro with anti-L3T4 antibody and complement, but not with anti-Lyt-2 antibody and complement. These results therefore demonstrate that L3T4+ T cells from mice chronically infected with T. cruzi can produce substances which interfere with the induction of DTH. This finding may help to identify the differential antigenic stimulatory requirement for the activation of the various subsets of T cells.

Effects of experimental acute pancreatitis in dogs on metabolism of lung surfactant phosphatidylcholine.

Acute haemorrhagic pancreatitis was produced in the dogs by transduodenal injection of autologous bile into the main pancreatic duct. There was no significant change in the activity of three regulatory enzymes of phosphatidylcholine biosynthesis (glycerophosphate acyltransferase, cytidyltransferase and cholinephosphotransferase) in lung; however, there was a 42% decrease in the amount of dipalmitoyl phosphatidylcholine (surfactant) in lung lavage due to acute pancreatitis. The decrease in lavage phospholipid content was associated with 5-fold increase in phospholipase A2 activity of lung lavage, and massive accumulation of osmiophilic spheroid structures in the alveolar space.

Adjuvant activity of saponin: antigen localization studies.

The adjuvant saponin potentiates the antibody response of mice to the antigen keyhole limpet hemocyanin (KLH). Studies using 125I-labelled KLH show that saponin significantly prolongs the retention of antigen at the subcutaneous injection site and also increases the amount reaching the spleen. Both these phenomena were associated with the inflammatory response to saponin and were markedly reduced following abolition of the inflammatory action of saponin by addition of cholesterol-containing liposomes. The adjuvant action of saponin was not modified by this treatment. Further evidence that neither antigen retention nor splenic localization is implicated in the adjuvant action of saponin for KLH is the demonstration that digitonin, which shares hemolytic and cholesterol binding activity with saponin, caused similar antigen retention and splenic localization but was not adjuvant active.

Immunization of marmosets with Trypanosoma cruzi cell surface glycoprotein (GP90).

Marmosets (Callithrix jacchus) have been immunized with a vaccine comprising a Trypanosoma cruzi 90K cell surface glycoprotein and the adjuvant saponin; a combination previously shown to be protective in mice. Immunization was by two s.c. injections one month apart and non-lethal challenge of homologous Y strain T. cruzi was given one month after the booster immunization. No anti-T. cruzi antibodies were detected after the first immunization but high levels developed after boosting. Immunization caused a significant decrease in the levels of acute phase blood parasitaemia, however, both immunized and control animals remained xenodiagnosis positive 60 weeks after infection. No ECG aberrations, histopathological lesions or anti-tissue antibodies were detected in infected marmosets.

The vaccine potential of cell surface glycoproteins from Trypanosoma cruzi.

An experimental Trypanosoma cruzi 90 kDa cell surface glycoprotein (GP90) vaccine, previously shown to be protective in mice is similarly effective in marmosets (Callithrix jacchus jacchus). Protection in the mouse is completely dependent on the adjuvant saponin and immunological studies confirm that GP90 is intrisically poorly immunogenic. Both specific antibody and cell mediated immunity are potentiated strongly by saponin and the resulting protective immunity is long lasting (six months). It is effective against the naturally infective, insect-metacyclic, form and a range of heterologous T. cruzi strains including a low mouse passage human isolate. Sterile immunity (that is, complete elimination of parasites) was not however, achieved. Evidence is presented that the levels of tissue damage associated with acute infection, as measured by production of auto anti-tissue immunoglobulins, are significantly reduced in GP90-immunized mice. These and other results are discussed in terms of the desired characteristics for vaccine use of T. cruzi antigens.

Restricted IgG isotype profiles in T. cruzi infected mice and Chagas' disease patients.

IgG2 was the predominant specific antibody isotype in mice chronically infected with Y strain Trypanosoma cruzi; IgG1 and IgG3 antibodies were absent or present only at very low levels. Isotype analyses of the acute phase of infection confirmed no early production of IgG1 or IgG3 and no failure in the switch from IgM to IgG. In vivo passive transfer studies of immune serum fractions showed protection to be associated only with the IgG2 isotype. A characteristic specific anti-T. cruzi IgG isotype profile (IgG1, IgG3, greater than IgG2, IgG4) was detected in a majority (39 of 50) of sera from Chagas' disease patients.

Direct chloride measurement of [4-36Cl]chlorobiphenyl and [4,4'-36Cl]dichlorobiphenyl dechlorination in the rat.

A reverse-phase HPLC system was used for the determination of inorganic chloride liberated in vivo from two biphenyl compounds in the rat. Oral administration of [4-36Cl]chlorobiphenyl resulted in a total yield of 27.6% of the original dose excreted over 10 d in the urine and included 1.7% of the dose as inorganic chloride. For [4,4'-36Cl]dichlorobiphenyl, the radioactivity of the original dose in the urine was 39.8% after 10 d, which included 11.5% of the dose as inorganic chloride. These results appear to represent the first direct determination of dechlorination by measurement of the inorganic chloride and suggest that biodechlorination plays a greater role in the metabolism of these compounds than previously expected.

In vitro dehalogenation of para-substituted aromatic halides in rat liver preparations.

The in vitro dehalogenation of a series of para-substituted halobenzenes was studied using HPLC separation followed by scintillation counting or neutron-activation analysis. Microsomal and cytosolic deiodination were established for iodobenzene substrates whose para-substituents were CO2H, CHO, NO2, OH, and C6H5 but not for para-iodobenzonitrile. A nonglutathione cytosolic deiodinase was only indicated with 4-iodobiphenyl as the substrate. In vitro dehalogenation could not be established for 4-bromobiphenyl using neutron-activation analysis.

Adjuvant requirements for protective immunization of mice using a Trypanosoma cruzi 90K cell surface glycoprotein.

A wide range of adjuvants including alhydrogel, saponin, Corynebacterium parvum, DDAB, Pfizer CP-20,961, oil adjuvants and several MDP analogues have been compared for their adjuvant activity in protecting mice against lethal Trypanosoma cruzi infection following immunization with a T. cruzi 90K cell surface glycoprotein. Only saponin was found to be effective. Promotion did not correlate with the ability to promote a particular Ig isotype; however, saponin was unique in its ability to promote cell-mediate immunity against the 90K glycoprotein.

A monoclonal antibody with specificity for Trypanosoma cruzi, central and peripheral neurones and glia.

Of 26 hybridomas secreting anti-Trypanosoma cruzi antibodies, two reacted with murine brain and spinal cord extracts but not other tissues. Both antibodies (5H7 and 3H3) had identical isotypes (IgM) and specificities as judged by western blotting. Antigens of molecular weight 58,000 and 37,000 in mouse brain and spinal cord, and 58,000 and 35,000 in T. cruzi were detected. Different tissue antigens were recognized by a previously reported T. cruzi mammalian neural tissue cross-reacting monoclonal antibody CE5; on immunofluorescence 5H7 stained some glia, and some central and peripheral neurones in rat tissue sections. Pooled sera from mice chronically infected with T. cruzi competed with binding of 5H7 to T. cruzi antigens.

75Se-methionine labelled Trypanosoma cruzi blood trypomastigotes: opsonization by chronic infection serum facilitates killing in spleen and liver.

Blood trypomastigote forms of Trypanosoma cruzi have been labelled with 75Se-methionine. Opsonization by sera from mice chronically infected with T. cruzi promoted their uptake by both liver and spleen. Opsonized parasites within spleens and livers were less infectious when transferred to normal recipients demonstrating in situ parasite killing by these organs.