A conserved TN8TCCT motif in the octapeptide-encoding region of Pax genes which has the potential to direct cytosine methylation

Our previous findings have shown that the developmental genes Pax7 and Pax3 are differentially methylated; the gene region that encodes the paired domain is hypomethylated, whereas the region that encodes the homeodomain is hypermethylated. For this reason, the known DNA sequence between the paired and homeoboxes was analysed for the presence of a conserved DNA motif to which a modifying protein could bind in order to direct the methylation or demethylation of surrounding gene sequences. The octapeptide-encoding region was found to contain several nucleotides that were highly conserved throughout the Pax gene family from phylogenetically distant species. The most conserved nucleotides are thought to comprise a motif TN8TCCT where N8=any combination of eight nucleotides. A conserved octapeptide-like-encoding sequence containing the TN8TCCT motif was also found in non-Pax genes of higher eukaryotes and in the non-coding strand of plants. Moreover, differential methylation seems to be associated with the presence of the TN8TCCT motif in p53 and the human oestrogen receptor genes. The presence of the TN8TCCT motif within an octapeptide-like-encoding sequence in human T-cell leukaemia virus type 1 might suggest that the putative recognition motif may have been introduced into various host genomes via some form of retroviral agent.     

The Glu318Gly mutation of the presenilin-1 gene does not necessarily cause Alzheimer's disease

In early-onset familial Alzheimer's disease (AD) pathogenic mutations have been found in the amyloid precursor protein (APP) gene and in the presenilin (PS)-1 and PS-2 genes. We screened for mutations in these genes in 20 patients with familial AD from the Finnish population. In addition, we sampled 41 sporadic AD patients and 59 controls to test for mutations identified in our familial AD cases. We detected an A-to-G transition in the PS-1 gene, resulting in a glutamic acid (Glu)-to-glycine (Gly) substitution at codon 318 in 2 familial and 2 sporadic AD patients. The Glu318Gly mutation has previously been reported to cause AD. We also found the Glu318Gly mutation in 4 healthy aged controls (range, 74-87 years). We thus conclude that the mutation is most likely a rare polymorphism not related to AD.     

Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions

The objective of this study was to determine if vascular cell adhesion molecule (VCAM-1), E-selectin, and P-selectin could selectively recruit leukocyte subpopulations, and whether this was affected by shear force or adhesion molecule concentration. Cover slips coated with purified adhesion molecules were incorporated into laminar flow chambers. Whole human blood was perfused for 5 minutes over these cover slips at relative shear forces of 2 to 40 dynes/cm2. Chasing the whole blood with buffer permitted visualization of leukocyte-substratum interactions. Leukocytes were observed to roll on and adhere to VCAM-1 at shears between 2 and 15 dynes/cm2. As assessed by cover slip staining, the majority of these cells were lymphocytes, but eosinophils, monocytes, and, surprisingly, neutrophils were also recruited, events inhibitable by anti-4-integrin antibody (HP1/2). Neutrophils were effectively recruited onto the selectins, with interactions occurring at shears as high as 30 and 40 dynes/cm2 for E- and P-selectin respectively. Eosinophils had high affinity for P- but not E-selectin. Mononuclear cells did not have high affinity for either selectin, but interacted avidly with VCAM-1. Antibodies against P-selectin (G1) and E-selectin (ES-1) completely blocked interactions on these substrates. Reducing the concentration of adhesion molecules did not appreciably change recruitment patterns except for VCAM-1, where neutrophils were no longer recruited. The novel use of whole blood in flow chambers shows a partial selectivity of selectins and VCAM-1 for certain subpopulations of leukocytes under varying physiologic shear conditions.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.     

Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Iq from the integron of a natural multiresistance plasmid

The nucleotide sequence of a newly identified amikacin resistance gene, aac(6')-Iq (551 bp), is reported. It has 68.4 and 94.4% homology with the aac(6')-Ia gene and the recently described aac(6')-Ip gene, respectively. Analysis of its flanking sequences indicated that it is in the first cassette of a class I integron and has an attC site (59-base element) 108 bp in length.     

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor

1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.     

Programmed cell death contributes to postnatal lung development

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.     

Insulin resistance precedes microalbuminuria in patients with insulin-dependent diabetes mellitus

BACKGROUND: Insulin resistance has been associated with hypertension and with renal complications in patients with type 1 diabetes mellitus. Causal relationships have not been fully explained. METHODS: We investigated whether insulin resistance precedes microalbuminuria by measuring insulin resistance with a euglycaemic clamp in combination with indirect calorimetry in 16 uncomplicated type 1 diabetic patients and in six healthy control subjects. The patients had over 10 year duration of diabetes, and were expected to experience either a complication-free or complicated disease course within the next few years. They have thereafter been followed for the development of microalbuminuria for 3 years. RESULTS: In a euglycaemic insulin clamp glucose disposal was lower in diabetic patients compared with control subjects (7.5 +/- 2.9 and 12.6 +/- 2.0 mg/kg LBM/min; P<0.002), mainly due to impaired glucose storage (4.3 +/- 2.3 vs 8.6 +/- 1.6 mg/kg LBM/min; P<0.001). Three years later seven IDDM patients had albumin excretion rate over 30 mg/24 h; glucose disposal (5.5 +/- 2.1 vs 9.0 +/- 2.2 mg/kg LBM/min; P<0.01) had been lower in patients who developed microalbuminuria compared with those who remained normoalbuminuric. CONCLUSIONS: Insulin resistance predicts the increment in urinary albumin excretion. Insulin resistance depends mainly on impaired glucose storage in uncomplicated IDDM.     

Glycoprotein IIb/IIIa receptor antagonists in the management of cardiovascular diseases

Recent studies of the effects of glycoprotein (GP) IIb/IIIa receptor antagonists on the clinical outcomes of patients with cardiovascular diseases are reviewed. The GP IIb/IIIa receptor antagonists studied include abciximab (a murine monoclonal antibody); eptifibatide (a synthetic peptide); and tirofiban, lamifiban, xemilofiban, sibrafiban, and lefradafiban (synthetic nonpeptides). A majority of clinical trials of GP IIb/IIIa receptor antagonists have been performed in patients with unstable angina or acute myocardial infarction and in patients undergoing percutaneous coronary interventions in whom an intracoronary thrombus may lead to ischemic complications. There is abundant evidence that GP IIb/IIIa receptor antagonists reduce the risk of death, acute myocardial infarction, and urgent revascularization procedures in high- and low-risk patients undergoing percutaneous coronary interventions. Abciximab remains the most studied of these agents in interventional settings. Data are accumulating on synthetic peptide and nonpeptide GP IIb/IIIa receptor antagonists that also demonstrate lower rates of death and ischemic complications in the treatment of acute coronary syndromes. In patients who have had a successful response to intravenous GP IIb/IIIa receptor antagonists, oral agents may represent an option for secondary prevention. Additional studies are required in order to determine further uses for these agents. A growing body of evidence supports the role of GP IIb/IIIa receptor antagonists in invasive and pharmacologic treatment approaches to acute coronary syndromes.