Lack of effect of ponsinomycin on the pharmacokinetics of nicoumalone enantiomers.

Pharmacokinetic interaction between ponsinomycin-nicoumalone was studied in six subjects who received an 8 mg oral dose of racemic nicoumalone alone and 4 days into an oral regimen of ponsinomycin 800 mg twice daily. The concentrations of R(+) and S(-)-nicoumalone in plasma were measured using a stereospecific h.p.l.c. assay. The disposition characteristics of nicoumalone enantiomers in the control phase of this study were similar to those reported previously with the exception of the data for one subject whose oral clearance for S(-)-nicoumalone was seven times lower than those in the other subjects. A statistically significant effect of ponsinomycin on the kinetics of R(+) and S(-)-nicoumalone was not demonstrated.     

Release of newly synthesized h-dopamine in the striatum: An adaptation of the push-pull cannula method to awake restrained and anesthetized rats

The release of newly synthesized ³H-dopamine (³H-DA) was measured in the rat striatum superfused, through a push-pull cannula, with a physiological medium enriched in ³H-tyrosine. The level of spontaneous ³H-DA release was dependent on the topographical localisation of the cannula in the striatum (anterior parts displayed higher levels than posterior ones) and on the anesthetic state (halothane anesthetized rats demonstrated higher levels than awake ones). Inhibition of DA inactivation processes by local application of benztropine (a DA reuptake inhibitor, 10⁻⁶ M) or by IV administration of pargyline (a MAO inhibitor, 100 mg/kg) enhanced the detectable outflow of ³H-DA from the striatum in both halothane anesthetized and awake rats. Local application of D-amphetamine (10⁻⁵ M) or acetylcholine (5 × 10⁻⁵ M) in the presence of eserine (5 × 10⁻⁵ M) evoked respectively a fivefold and a 30% increase in spontaneous ³H-DA release in halothane anesthetized rats. Inhibition of the firing of dopaminergic neurons by IV injection of gamma-hydroxybutyrate (400 mg/kg) produced a 30% decrease in striatal ³H-DA release. The present results demonstrate that the push-pull cannula method is suitable for the study of DA release in both the anesthetized and the awake rat.     

Organization of point contacts in neuronal growth cones

Growth cones from rat dorsal root ganglia plated on laminin contain integrin clusters over the entire growth cone surface, and growth cones make transient adhesions at sites called point contacts. We examined, by immunocytochemistry and confocal microscopy, the composition and distribution of point contacts in neuronal growth cones. Vinculin was concentrated in the central domain of growth cones and at the tips of filopodia. Vinculin was specifically associated with integrin clusters at the membrane-substrate interface and thus marked point contacts. The cytoskeletal proteins paxillin and talin colocalized with beta1 integrin in a subpopulation of clusters restricted to the central domain of the growth cone and to the tips of filopodia. The neuron-specific kinase, FAK+ also distributed with the vinculin-positive clusters. The Rho family proteins RhoA, RhoB, and Cdc42 were present in growth cones, and a few Rho clusters were colocalized with vinculin. Examination of proteins resistant to detergent extraction in PC12 cells confirmed the retention of beta1 integrin, paxillin, talin, and vinculin with the cytoskeleton. Moreover, we detected FAK+ and RhoA in the detergent-resistant cytoskeleton, supporting their distribution to point contacts. Our observations indicate that two types of integrin clusters are present in growth cones: those associated with vinculin at the cell substratum interface, and those not associated with vinculin. Point contacts are mature adhesion sites defined by the presence of both beta1 integrin and vinculin, and they are associated with signaling proteins.     

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.     

Reduction of graft failure by a monoclonal antibody (anti-LFA-1 CD11a) after HLA nonidentical bone marrow transplantation in children with immunodeficiencies, osteopetrosis, and Fanconi's anemia: a European Group for Immunodeficiency/European Group for Bone Marrow Transplantation report

Forty-six infants and children suffering from either inherited immunodeficiency disorders (Wiskott-Aldrich syndrome, functional T-cell immunodeficiency with or without HLA class II expression deficiency), malignant osteopetrosis, or Fanconi's anemia received HLA-nonidentical bone marrow transplantation (BMT) from related donors. Bone marrow was T-cell depleted to reduce the risk of graft-versus-host disease (GVHD). To prevent graft failure, a mouse monoclonal antibody specific for the CD11a-lymphocyte function-associated antigen 1 (LFA-1) molecule was infused into the patients. Eleven patients received five infusions of 0.1 mg/kg every other day from day -3 to +5. Thirty-five patients received 0.2 mg/kg daily from day -3 to +6. The overall sustained engraftment rate was 72% instead of 26.1% in a historical control group of 24 patients similarly treated except for the infusion of the anti-LFA-1 antibody. No late rejection occurred. The T-cell depletion method (E-rosetting or Campath IM plus complement) resulted in different rate of engraftment (83.3% v 57.9%, respectively, P = .05). Engraftment rate was slightly but not significantly influenced by the degree of HLA incompatibility between donor and recipient. Acute GVHD of grade II or more occurred in 35.5% of the patients and the rate of chronic GVHD was 12.9%. The overall actuarial survival rate with a functional graft is 47.3% with a mean follow-up of 28.0 months for patients with immunodeficiency and osteopetrosis, while none of the four patients with Fanconi's anemia survived. The development of full T-cell functions took on the average 6 months and of full B-cell functions 10 months. Significant infectious problems developed in the majority of the patients during the posttransplant course. Epstein-Barr virus-induced B-cell proliferative syndromes were observed in seven patients, six of whom had Wiskott-Aldrich syndrome. Correction of immunodeficiency was comparable in terms of kinetics and quality with that observed in patients with severe combined immunodeficiency undergoing HLA-nonidentical BMT. Correction of osteopetrosis appears not to be different from what has been observed after HLA-identical BMT. The in vivo use of an anti-CD11a-LFA-1 antibody as an additional immunosuppressive therapy in HLA-nonidentical BMT may thus promote engraftment and survival with correction of the primary disease in a significant number of patients with life-threatening immunodeficiency and osteopetrosis, but not with Fanconi's anemia.     

Dissociation of hypotensive and renal hemodynamic effects of an angiotensin converting enzyme inhibitor in insulin-dependent diabetic patients with incipient nephropathy

The mechanism of action of angiotensin converting enzyme (ACE) inhibitors on urinary albumin excretion (UAE) in diabetics is controversial. In order to dissociate the hypotensive and intrarenal effects, 16 insulin-dependant diabetics with permanent microalbuminuria (30-300 mg/24 h) without hypertension were given Ramipril, a long acting ACE inhibitor, at hypotensive (treatment A 5 mg/day; N = 8) and at sub-hypotensive doses (treatment B, 1.25 mg/day; N = 8) over a 6 week period in parallel double-blind study. Blood pressure, UAE, glomerular filtration renal blood flow (continuous 125I-Iodothalamate + 131I-Hippurate infusion) and converting enzyme activity (Liebermann's method), before and after treatment. In treatment group A, the blood pressure fell from 133 +/- 5/79 +/- 4 (mean +/- SE) to 125 +/- 4/77 +/- 2 mmHg (p less than 0.05 for systolic blood pressure) whereas it remained stable in treatment group B (132 +/- 7/79 +/- 4 to 128 +/- 5/80 +/- 4 mmHg). The UAE decreased in both groups: group A from an average of 74 (40-198) to 47 (5-202) mg/24 h (p = 0.07; group B, from an average of 77 (50-136) to 19 (15-120) mg/24 h (p less than 0.005), as did ACE activity: group A from 332 +/- 44 to 163 +/- 33 iu/l (p less than 0.004), group B from 423 +/- 39 to 191 +/- 28 iu/l (p less than 10-4).(ABSTRACT TRUNCATED AT 250 WORDS)