Non-cholinergic effects of acetylcholinesterase in the substantia nigra: a possible role for an ATP-sensitive potassium channel

Summary Within the substantia nigra acetylcholinesterase has non-cholinergic actions that can be demonstrated at both behavioural and cellular levels: the aim of this study was, thus, to explore, in the in vitro guinea pig substantia nigra, the ionic mechanisms which mediate these non-classical phenomena. Acetylcholinesterase had a reversible hyperpolarizing action, via an opening of potassium channels, on a selective population of nigral neurons. These neurons could be identified by an ability to generate bursts of action potentials and by a sensitivity to either amphetamine or to a reduction of glucose in the perfusing medium. The acetylcholinesterase-induced hyperpolarization could not be attributed to a contaminant in the exogenous solution, since a highly purified preparation was even more potent. Furthermore, enzymatic action of any kind could be eliminated as boiled acetylcholinesterase was equally efficacious. The effect of acetylcholinesterase was not subject to tachyphylaxis and was resistant to blockade of potassium channels with tetraethylammonium: since both these phenomena are features of the D₂ autoreceptor for dopamine within the substantia nigra, it seems unlikely that acetylcholinesterase is operating on the same target as dendritically released local dopamine. On the other hand, the actions of acetylcholinesterase were enhanced by low glucose and blocked by the sulfonylurea, tolbutamide. These results strongly suggest that acetylcholinesterase can exert a nonenzymatic action and that this action, in the substantia nigra, is mediated by an ATP-sensitive potassium channel.     

Generating compensation designs for tangential breast irradiation with artificial neural networks

In this paper we discuss a study comparing an algorithm implemented clinically to design intensity-modulated fields with two artificial neural networks (ANNs) trained to design the same fields. The purpose of the algorithm is to produce compensation for tangential breast radiotherapy in order to improve dose homogeneity. This was achieved by creating intensity-modulated fields to supplement standard wedged fields. Portal image data were used to create thickness maps of the medial and lateral fields, which in turn were used to design the wedged and intensity-modulated fields. The ANNs were developed to design the intensity-modulated fields from the portal image data and corresponding fluence map alone. One used localized groups of portal image pixels related to the fluence map (method 2), and the other used a one-to-one mapping between spatially corresponding pixels (method 3). A dosimetric comparison of the methods was performed by calculating the overall dose distribution. The volume of tissue outside the dose range 95-105% was used to assess dose homogeneity. The average volume outside 95-105%, averaged over 80 cases, was shown to be 2.3% for the algorithm, whilst average values of 9.9% and 13.5% were obtained for methods 2 and 3, respectively. The results of this study demonstrate the ability of an ANN to learn the general shape of compensation required and explore the use of image-based ANNs in the design of intensity-modulated fields.     

An optimization algorithm that incorporates IMRT delivery constraints

An intensity-modulated beam optimization algorithm is presented which incorporates the delivery constraints into the optimization cycle. The optimization algorithm is based on the quasi-Newton method of iteratively solving minimization problems. The developed algorithm iteratively corrects the incident, pencil-beam-like, fluence to incorporate the delivery constraints. In the present study, the goal of the optimization algorithm is to achieve the best deliverable radiotherapy plan, subject to the constraints of the delivery technique described by a leaf-sequencing algorithm being applied concurrently. In general, if they are applied after, rather than during, the optimization cycle, the delivery constraints associated with the IMRT technique can produce local variations up to 6% in the 'optimized' dose (i.e., distribution without applied constraints) and reduce the degree of conformity, of the dose, to the PTV region. The optimization method has been applied to three IMRT delivery techniques: dynamic multileaf (DMLC), multiple-static-field (MSF) and slice-by-slice tomotherapy (NOMOS MIMiC). The beam profiles were generated for a prostate tumour with organs at risk being the rectum, bladder and femoral heads. The optimization method described was shown to generate optimum and deliverable IMRT plans for these three delivery techniques. In the case of the DMLC and MSF the optimization converged within 3-5 iterations to a mean PTV dose of 69.60 +/- 1.34 Gy and 69.71 +/- 1.34 Gy, respectively, while for NOMOS MIMiC approximately 10 iterations were needed to obtain 69.68 +/- 1.55 Gy. In addition to this, the IMRT optimization also yielded optimum fluence profiles when clustering was performed concurrently with the leaf-sequencer. An optimum between 8 and 15 clusters of equal fluence 'intensity' was shown to establish the best compromise between the number of fluence levels and the PTV dose coverage.     

Concepts for shuttling multileaf collimators for intensity-modulated radiation therapy

Shuttling multileaf collimators (SMLCs) can increase the MU efficiency of intensity-modulated radiation therapy compared with the multiple-static-field (MSF-MLC) technique or dynamic MLC (DMLC) technique with conventional MLCs. In a previous paper (Phys. Med. Biol. 45 3343-58) a particular SMLC was shown, for highly modulated intensity distributions, to increase the MU efficiency compared with the MSF-MLC technique. In this companion paper, two new arrangements similar to that described in the earlier paper, but with less mechanical complexity, are shown to be constructionally simpler but less MU efficient. Additionally another new concept of SMLC is shown which also increases the MU efficiency compared with the MSF-MLC technique and often improves the MU efficiency compared with the previously reported SMLC for highly modulated intensity distributions. It also leads to zero tongue-and-groove underdose in the direction orthogonal to that of the shuttling elements (so-called across-the-rows).     

Assessing lymphocyte functions in neonates for revealing abnormal prenatal development of the immune system

Because it is difficult to assess prenatally induced functional deficits of the human immune system, we developed an ex vivo method for differentiation and maturation of peripheral lymphocytes of newborn, preferentially using umbilical cord blood. Many lymphocyte subsets of newborn infants are "immature" with respect to defined surface receptors. An example of such an immaturity is the almost complete lack of "memory"-type helper T cells (also designated as helper-inducer cells), characterized by expressing the surface receptors: CD4(+)CD45R0(+)CD45RA(-)CD29(high). On the other hand, umbilical cord blood contains many "naive"-type helper T cells (often designated as suppressor-inducer cells), with the receptors: CD4(+)CD45R0(-)CD45RA(+)CD29(low). In this report, we demonstrate that the immature helper lymphocyte population of umbilical cord blood is capable of differentiating to mature cells following stimulation with pokeweed mitogen (PWM) and other stimulants ex vivo. The obtained receptor pattern is virtually indistinguishable from the one observed on the mature cells of adults. Such an extensive differentiation can only be achieved with cells of newborns. As intermediates during differentiation in culture, CD45R0(+)CD45RA(+) cells may be observed which are rather rare in vivo. Additionally, the appearance of several activation (CD25, CD69, HLA-DR) and adhesion (CD11a, CD11b, CD11c, CD18, CD49b, CD49d, CD54) receptors on CD4 cells were analyzed. With this model system evidence for the sequence of events during differentiation and maturation may be obtained. This ex vivo-model is capable of studying the capacity of lymphocytes for differentiation and activation processes barely accessible in vivo. It may also be expected to represent an interesting tool for measuring the capacity for maturation and differentiation in the blood of children of different ages under normal and pathological conditions ex vivo. In addition, substance-induced effects may be studied in vitro with this approach on immature cells from newborn, or infants during culturing. Teratogenesis Carcinog. Mutagen. 20:171-193, 2000.     

The fragile (X) syndrome: the mutation problem

In an attempt to understand the nature of the mutational event leading to the fra(X) syndrome, we have searched for sporadic cases in 3 populations: affected males, affected females, and non-affected transmitting females. In all 3 populations there was a dearth of isolated cases, and the reasons for this are discussed.     

The Reliability of Arousal Threshold During Sleep

Thirty-five subjects from two independent studies were awakened at EEG-defined periods during the night with 1000 Hz ascending tone series. Awakenings were made five to eight times per night during stage 2, stage 4, or REM sleep over a series of nights in good and poor sleepers. Reliability was assessed within stage, within night, between stages, and between nights. Good and poor sleepers did not differ in either depth of sleep or reliability of arousal threshold and were thus pooled in the analyses. From night to night, the most consistency was seen in stage 4 (r=.74), although REM reliability (r?₁= .49) and stage 2 reliability (r?₁= .50 and r?₁= .69 in the two respective studies) estimates were also greater than zero. Early sleep onset and morning arousals were more variable. Reliability estimates on arousal thresholds taken within the same night for stage 2 were r= .64 and r?₁= .77 for the two studies and r= .96 for REM. The depth of sleep was not correlated with awake auditory threshold. It was concluded that five or six carefully placed arousals could give a good estimate of an individual's usual arousal threshold.     

Evaluation of Streptococcus pneumoniae Type XIV Opsonins by Phagocytosis-Associated Chemiluminescence and a Bactericidal Assay

The relative roles of serum factors required for opsonization of type XIV Streptococcus pneumoniae were investigated by means of luminol-enhanced chemiluminescence (CL), bactericidal, and immunofluorescence assays employing adult sera containing high (>1,000 ng of antibody nitrogen per ml) or low (<200 ng of antibody nitrogen per ml) antibody concentrations as determined by radioimmunoassay. Specific antibody concentration correlated directly with both total and heat-labile CL activity (P < 0.005) and with the bactericidal index (P < 0.05) at a serum concentration of 10%. The importance of specific antibody as an opsonin was confirmed by the abolition of CL activity and immunoglobulin immunofluorescence observed after absorption of heated sera with type XIV pneumococcal cells and by the dose response in CL and bactericidal activity observed with the addition of immunoglobulin G to hypogammaglobulinemic serum. A role for the classical complement pathway in opsonization was indicated by significantly greater CL integrals for high-antibody sera than for low-antibody sera depleted of factor D and by the bactericidal activity noted for untreated, but not magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid-chelated low-antibody sera. The alternative pathway contributed more than half of the CL activity of both high- and low-antibody sera. However, after magnesium ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid chelation, only sera with high antibody concentrations or agammaglobulinemic serum reconstituted with immunoglobulin G with high specific antibody levels supported significant bactericidal activity. Therefore, type-specific antibody and complement promote opsonization of type XIV S. pneumoniae, and this may occur via either complement pathway. These results suggest that CL is a suitable tool to delineate serum factors and their contribution to opsonization, but results must be related to other functional assays.     

The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.