Thrombin receptor (PAR-1) antagonists. Heterocycle-based peptidomimetics of the SFLLR agonist motif

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.     

Telomere length regulation in mice is linked to a novel chromosome locus

Little is known about the mechanisms that regulate species-specific telomere length, particularly in mammalian species. The genetic regulation of telomere length was therefore investigated by using two inter-fertile species of mice, which differ in their telomere length. Mus musculus (telomere length >25 kb) and Mus spretus (telomere length 5-15 kb) were used to generate F1 crosses and reciprocal backcrosses, which were then analyzed for regulation of telomere length. This analysis indicated that a dominant and trans-acting mechanism exists capable of extensive elongation of telomeres in somatic cells after fusion of parental germline cells with discrepant telomere lengths. A genome wide screen of interspecific crosses, using M. spretus as the recurrent parent, identified a 5-centimorgan region on distal chromosome 2 that predominantly controls the observed species-specific telomere length regulation. This locus is distinct from candidate genes encoding known telomere-binding proteins or telomerase components. These results demonstrate that an unidentified gene(s) mapped to distal chromosome 2 regulates telomere length in the mouse.     

Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.     

Pancreatic beta-cell regeneration after 48-h glucose infusion in mildly diabetic rats is not correlated with functional improvement

We investigated the effect of glucose infusion on beta-cell regeneration in rats made mildly diabetic by a single injection of low dosage (35 mg/kg) streptozotocin (STZ). Nondiabetic (ND) and STZ rats were submitted to a 48-h glucose infusion (hyperglycemia approximately 22 mmol/l in both groups: ND and STZ hyperglycemic-hyperinsulinemic [ND HG-HI and STZ HG-HI rats]). Before infusion, beta-cell mass was 65% lower in STZ rats than in ND rats (2.0 +/- 0.02 vs. 5.5 +/- 0.6 mg), 1.6-fold increased in ND HG-HI rats (8.7 +/- 1.7 mg), and 2.7-fold increased in STZ HG-HI rats (5.4 +/- 0.9 mg). In ND HG-HI rats, beta-cell enlargement was related to an increase in beta-cell responsiveness to nutrient secretagogues both in vivo and in vitro, whereas in STZ HG-HI rats, no significant improvement in insulin secretion could be noticed. To determine the respective role of hyperglycemia and hyperinsulinemia on beta-cell area changes, ND and STZ rats were submitted to a 48-h hyperinsulinemic-euglycemic clamp. No modification of beta-cell mass was detected in either group. In conclusion, 48-h superimposed hyperglycemia was enough to restore beta-cell mass previously reduced by STZ injection. This effect seemed to be due to hyperglycemia rather than hyperinsulinemia alone. The data stress the dissociation between beta-cell regeneration and improvement in islet function in diabetic rats. Our model seems suitable for studying factors that can improve the plasticity and function of the pancreas in NIDDM.     

Kinetics of the unfolding-folding transition of Bacillus subtilis levansucrase precursor

The reversible folding-unfolding transition of mature and precursor forms of Bacillus subtilis levansucrase were compared under physiological conditions of pH and temperature. The time constant of the folding reaction was not modified by the presence of the signal sequence and the precursor in the native form was slightly more resistant to the denaturing action of urea. However, the folding pathway could be different for each protein since a domain of the mature levansucrase underwent an independent transition which is not observed during the renaturation process of prelevansucrase.     

Medical-resource use for suspected tuberculosis in a New York City hospital

OBJECTIVE: To compare resource use by diagnostic outcome among hospital admissions during which tuberculosis (TB) was suspected. DESIGN: Retrospective study based on chart review and microbiology laboratory data. SETTING: The department of medicine in a municipal hospital serving central Brooklyn, New York. PARTICIPANTS: We identified all adult admissions in 1993 during which TB was suspected. We assigned each admission to one of four mutually exclusive groups defined by the results of microbiological tests (acid-fast bacilli [AFB] smear and culture): culture-positive and smear-positive (C+S+); culture-positive and smear-negative (C+S-); culture-negative and smear-positive (C-S+); or culture-negative and smear-negative (C-S-). Each admission was divided into two separate periods to which the utilization of medical resources was assigned: the diagnostic and the postdiagnostic periods, which were separated by the date of receipt of the first definitive culture report. RESULTS: Data on 519 admissions (93 C+S+; 57 C+S-; 30 C-S+; and 339 C-S-) were analyzed. Although C+S+ were more likely than other groups to have an admitting diagnosis of TB, approximately one quarter of the admissions without TB (C-S+, C-S-) were admitted with the principal diagnosis of TB. For the four groups, C+S+, C+S-, C-S+, and C-S-, the respective rates of TB isolation and anti-TB treatment, and median lengths of isolation were 98%, 87%, and 34 days; 74%, 74%, and 7 days; 83%, 83%, and 15 days; and 44%, 29%, and 0 days. During the diagnostic period, the rate and length of isolation were similar in the AFB-smear-positive groups (C+S+ and C-S+). We estimated that admissions without culture-proven TB (C-S+ and C-S-) accounted for 3,174 (36%) of the 8,712 days of TB isolation expended and for 65% of the 16,671 days of anti-TB treatment. The vast majority of this resource consumption (2,737 [86%] of 3,174 days of isolation) occurred during the diagnostic period before a definitive culture result was known. CONCLUSIONS: Our results suggest that prolonged diagnostic uncertainty and misclassification of cases due to false-positive and false-negative smears are associated with substantial medical-resource consumption. New diagnostic modalities that reduce the period of diagnostic uncertainty could reduce the utilization of resources later found to be unnecessary.     

Nonsyndromal craniosynostosis: longitudinal outcome following cranio-orbital reconstruction in infancy

This paper presents a prospective longitudinal outcome study on patients with nonsyndromal craniosynostosis who were treated with the contemporary craniofacial surgical techniques of suture release, cranial decompression, and cranial and orbital reconstruction and reshaping in infancy. Diagnosis, surgical treatment, and long-term results and complications are reviewed. Preoperative and long-term postoperative intracranial volumes in these patients were evaluated and compared with age and gender match controls throughout the period of the study. From July 1, 1990, to July 1, 1994, 25 patients with isolated nonsyndromal craniosynostosis underwent surgery of the deformity. Eight patients were excluded from the study based on incomplete postoperative computed tomography (CT) records. Of the 17 patients with long-term computerized records, 11 were boys and 6 were girls. The nonsyndromal craniosynostosis patients in this study include six with bilateral coronal craniosynostosis, six with unilateral coronal craniosynostosis, four with sagittal craniosynostosis, and one with metopic craniosynostosis. The average age at the time of surgery for all patients was 9 months, and the average age at the time of the latest follow-up CT scan for all patients in the study was 3.5 years. There were no perioperative complications in this series of patients including no bleeding, no infection, no wound healing complications, and no mortality. Bony fixation included a combination of wire osteosynthesis and rigid microfixation. All patients had only one surgical procedure for the correction of their deformity. Evaluation of both preoperative and long-term postoperative intracranial volume measurements in this series of patients revealed that these volume measurements were comparable with the gender match control groups at all ages throughout the study. The significance of these findings for this longitudinal outcome study is discussed.     

Antitumour polycyclic acridines. Part 4. Physico-chemical studies on the interactions between DNA and novel tetracyclic acridine derivatives

The non-covalent interactions between a series of new tetracyclic acridine derivatives (5-11) and DNA have been studied by spectrophotometric analysis, fluorescences quenching, thermal denaturation, and circular and linear dichroism. In order to compare the extent of the DNA binding by compounds 5-11 in their neutral and cationic forms, all experiments were conducted at pH 7.4 (physiological pH) and 5.0. The results indicated that compounds 5-11 are strong DNA-binding ligands with DNA affinities comparable to that of m-AMSA (1) or even higher. They showed a stronger DNA binding activity at pH 5.0 as a result of the N-protonation of the pyridoacridine aromatic chromophore. Ethidium-DNA fluorescence assays showed an A-T base pair preference of the binding distinguishing these novel compounds from simple acridines which show a slight G-C base pair preference. Circular and linear dichroism studies indicated that the drugs bind to DNA by undergoing intercalation inside the duplex macromolecule at high DNA:drug ratios and revealed alternative binding modes at low DNA:drug ratios.