NMDA-receptor contribution to spinal nociceptive reflexes: influence of stimulus parameters and of preparatory surgery

The contribution of NMDA receptors to nociceptive reflexes has been assessed both in awake rats and in electrophysiological tests on alpha-chloralose anaesthetized spinalized rats prepared with different degrees of surgery. Single motor unit activity was recorded in response to alternating noxious mechanical and electrical stimuli applied to one hindpaw, and the results compared with paw pressure withdrawal reflexes in awake rats. There was little contribution by NMDA receptors to nociceptive paw pinch responses either in awake rats or in rats prepared with minimal surgery, but following extensive lumbar surgery the contribution increased significantly to a level similar to that seen in the wind-up component of responses elicited by electrical stimulation. Surgery therefore has effects several segments from the sensory input that it generates. It enhances the NMDA receptor contribution in responses to some but not all types of afferent input.     

Biofeedback training in treatment of childhood constipation: a randomised controlled study

BACKGROUND: Because abnormal defaecation dynamics, which can be modified by biofeedback, are considered to be the underlying problem in constipation, biofeedback training may be a useful treatment for constipation. This treatment has mainly been studied in uncontrolled trials. We evaluated defaecation dynamics and clinical outcome in chronically constipated children in a randomised study comparing conventional treatment and conventional treatment with biofeedback training. METHODS: Patients, 5 to 16 years old, were referred to the Academic Medical Center in Amsterdam by general practitioners, school doctors, paediatricians, and psychiatrists. They had to fulfil at least two of four criteria for paediatric constipation and were included if they had been treated medically for at least one month before randomisation. Patients had a medical history, abdominal and rectal examination, and anorectal manometry at the start and end of the 6-week intervention period. The conventional group received laxative treatment with additional dietary advice, toilet training, and maintenance of a diary of bowel habits. The biofeedback group received the same conventional treatment and additionally five biofeedback training sessions. During the first 3 weeks, patients visited the outpatient clinic weekly; two subsequent visits were twice monthly. FINDINGS: 94 patients were randomised to conventional treatment (CT) and 98 to conventional treatment with additional biofeedback training (CT+BF). Normal defaecation dynamics increased in the CT group from 41% to 52% (not significant) and in the CT+BF group from 38% to 86% (p = 0.001). At 6 weeks, more patients in the CT+BF group showed normal defaecation dynamics, compared to the CT group (p < 0.001). This result was unaltered by controlling for baseline status in a logistic regression model. At 1 year, successful treatment (defaecation frequency > or = 3/week, soiling and/or encopresis < 2/month, and no laxatives) was accomplished in 59% of the CT and 50% of the CT+BF group (p = 0.24). The results were maintained after 1 1/2 years follow-up. No association was found between achievement of normal defaecation dynamics and clinical outcome. INTERPRETATION: Additional biofeedback training compared to conventional therapy did not result in higher success rates in chronically constipated children. Furthermore, achievement of normal defaecation dynamics was not associated with success: abnormal defaecation dynamics seem not to play a crucial role in the pathogenesis of childhood constipation. Intensive conventional laxative treatment should remain the first choice in chronically constipated children.     

Liquid levodopa/carbidopa produces significant improvement in motor function without dyskinesia exacerbation

We performed the first double-blind, crossover comparison between levodopa/carbidopa (LD/CD) in optimized liquid versus tablet doses to measure plasma LD levels and relative effects on disabilities (motor function, fluctuations, and dyskinesias) in patients with Parkinson's disease. Twenty-three subjects with motor fluctuations were optimized with open-label LD/CD tablets and liquid. In a double-dummy design, patients randomly received 2 weeks of liquid and 2 weeks of tablet LD/CD. Twice during each arm, we evaluated patients hourly 9 AM to 4 PM with the use of plasma LD levels, the Unified Parkinson's Disease Rating Scale, a dyskinesia rating scale, and on-off ratings. Patients receiving liquid LD/CD ingested significantly higher doses and had significantly improved motor function and total on time, without an increase in dyskinesia severity. The number of motor fluctuations in the two phases was not significantly different. LD levels and variability were also equivalent with the two formulations. At optimized dosing, liquid LD/CD offers a means to significantly improve motor disability in patients with Parkinson's disease without exacerbating dyskinesia.     

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.     

Factor V (Arg 506-->Gln) mutation in young survivors of myocardial infarction

Many young patients with venous thromboembolic disease are partially resistant to the anticoagulant action of activated protein C as a result of factor V (Arg 506 --> Gln) mutation. The frequency of this mutation in young patients with arterial thrombotic diseases, such as myocardial infarction, is less well established. We studied 100 young patients with myocardial infarction and 100 age- and sex-matched controls. One patient (1%; 95% CL 0.05-6.2) and two controls (2%; 95% CL 0.3-7.7) were heterozygotes for the mutation; there was no homozygote in either group. Hence, premature myocardial infarction is not associated with heterozygosity for factor V (Arg 506 --> Gln) mutation.     

Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination

Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.     

Modeling, chemistry, and biology of the benzolactam analogues of indolactam V (ILV). 2. Identification of the binding site of the benzolactams in the CRD2 activator-binding domain of PKCdelta and discovery of an ILV analogue of improved isozyme selectivity

Protein kinase C (PKC) is a complex enzyme system comprised of at least 11 isozymes that serves to mediate numerous extracellular signals which generate lipid second messengers. The discovery of isozyme-selective activators and inhibitors (modulators) of PKC is crucial to ascertaining the role of the individual isozymes in physiological and pathophysiological processes and to manipulating their function. The discovery of such small molecule modulators of PKC is at present a largely unmet pharmacological need. Herein we detail our modeling studies which reveal how the natural product indolactam V (ILV) and its 8-membered ring analogue, the benzolactam 15, bind to the CRD2 activator domain of PKC. These modeling studies reveal that not all PKC ligands possess a common pharmacophore, and further suggest an important role of specific hydrophobic contacts in the PKC-ligand interaction. The modeling studies find strong experimental support from mutagenesis studies on PKC alpha that reveal the crucial role played by the residues proline 11, leucine 20, leucine 24, and glycine 27. Next, we describe the synthesis of two 8-substituted benzolactams starting from L-phenylalanine and characterize their isozyme selectivity; one of the two benzolactams exhibits improved isozyme selectivity relative to the n-octyl-ILV. Lastly, we report inhibition of cellular proliferation of two different breast carcinoma cell lines by the benzolactam 5 and show that the compound preferentially down-regulates PKCbeta in both cell lines.