Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ～20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.     

Targeting the Neurokinin Receptor 1 with Aprepitant: A Novel Antipruritic Strategy

Background

Chronic pruritus is a global clinical problem with a high impact on the quality of life and lack of specific therapies. It is an excruciating and frequent symptom of e.g. uncurable renal, liver and skin diseases which often does not respond to conventional treatment with e.g. antihistamines. Therefore antipruritic therapies which target physiological mechanisms of pruritus need to be developed. Substance P (SP) is a major mediator of pruritus. As it binds to the neurokinin receptor 1 (NKR1), we evaluated if the application of a NKR1 antagonist would significantly decrease chronic pruritus.

Methods and Findings

Twenty hitherto untreatable patients with chronic pruritus (12 female, 8 male; mean age, 66.7 years) were treated with the NKR1 antagonist aprepitant 80 mg for one week. 16 of 20 patients (80%) experienced a considerable reduction of itch intensity, as assessed by the visual analog scale (VAS, range 0 to 10). Considering all patients, the mean value of pruritus intensity was significantly reduced from 8.4 VAS points (SD +/−1.7) before treatment to 4.9 VAS points (SD +/−3.2) (p<0.001, CI 1.913–5.187). Patients with dermatological diseases (e.g. atopic diathesis, prurigo nodularis) had the best profit from the treatment. Side-effects were mild (nausea, vertigo, and drowsiness) and only occurred in three patients.

Conclusions

The high response rate in patients with therapy refractory pruritus suggests that the NKR1 antagonist aprepitant may indeed exhibit antipruritic effects and may present a novel, effective treatment strategy based on pathophysiology of chronic pruritus. The results are promising enough to warrant confirming the efficacy of NKR1 antagonists in a randomized, controlled clinical trial.     

Differential effects of painful and non-painful stimulation on tactile processing in fibromyalgia syndrome and subjects with masochistic behaviour

Background

In healthy subjects repeated tactile stimulation in a conditioning test stimulation paradigm yields attenuation of primary (S1) and secondary (S2) somatosensory cortical activation, whereas a preceding painful stimulus results in facilitation.

Methodology/Principal Findings

Since previous data suggest that cognitive processes might affect somatosensory processing in S1, the present study aims at investigating to what extent cortical reactivity is altered by the subjective estimation of pain. To this end, the effect of painful and tactile stimulation on processing of subsequently applied tactile stimuli was investigated in patients with fibromyalgia syndrome (FMS) and in subjects with masochistic behaviour (MB) by means of a 122-channel whole-head magnetoencephalography (MEG) system. Ten patients fulfilling the criteria for the diagnosis of FMS, 10 subjects with MB and 20 control subjects matched with respect to age, gender and handedness participated in the present study. Tactile or brief painful cutaneous laser stimuli were applied as conditioning stimulus (CS) followed by a tactile test stimulus (TS) 500 ms later. While in FMS patients significant attenuation following conditioning tactile stimulation was evident, no facilitation following painful stimulation was found. By contrast, in subjects with MB no attenuation but significant facilitation occurred. Attenuation as well as facilitation applied to cortical responses occurring at about 70 ms but not to early S1 or S2 responses. Additionally, in FMS patients the amount of attenuation was inversely correlated with catastrophizing tendency.

Conclusion

The present results imply altered cortical reactivity of the primary somatosensory cortex in FMS patients and MB possibly reflecting differences of individual pain experience.     

Nature of the periplastidial pathway of starch synthesis in the cryptophyte Guillardia theta

The nature of the periplastidial pathway of starch biosynthesis was investigated with the model cryptophyte Guillardia theta. The storage polysaccharide granules were shown to be composed of both amylose and amylopectin fractions with a chain length distribution and crystalline organization very similar to those of starch from green algae and land plants. Most starch granules displayed a shape consistent with biosynthesis occurring around the pyrenoid through the rhodoplast membranes. A protein with significant similarity to the amylose-synthesizing granule-bound starch synthase 1 from green plants was found as the major polypeptide bound to the polysaccharide matrix. N-terminal sequencing of the mature protein proved that the precursor protein carries a nonfunctional transit peptide in its bipartite topogenic signal sequence which is cleaved without yielding transport of the enzyme across the two inner plastid membranes. The enzyme was shown to display similar affinities for ADP and UDP-glucose, while the V(max) measured with UDP-glucose was twofold higher. The granule-bound starch synthase from Guillardia theta was demonstrated to be responsible for the synthesis of long glucan chains and therefore to be the functional equivalent of the amylose-synthesizing enzyme of green plants. Preliminary characterization of the starch pathway suggests that Guillardia theta utilizes a UDP-glucose-based pathway to synthesize starch.     

Chronotropic response of cultured neonatal rat ventricular myocytes to short-term fluid shear

Ventricular myocytes are continuously exposed to fluid shear in vivo by relative movement of laminar sheets and adjacent cells. Preliminary observations have shown that neonatal myocytes respond to fluid shear by increasing their beating rate, which could have an arrhythmogenic effect under elevated shear conditions. The objective of this study is to investigate the characteristics of the fluid shear response in cultured myocytes and to study selected potential mechanisms. Cultured neonatal rat ventricular myocytes that were spontaneously beating were subjected to low shear rates (5-50/s) in a fluid flow chamber using standard culture medium. The beating rate was measured from digital microscopic recordings. The myocytes reacted to low shear rates by a graded and reversible increase in their spontaneous beating rate of up to 500%. The response to shear was substantially attenuated in the presence of the beta-adrenergic agonist isoproterenol (by 86+/-8%), as well as after incubation with integrin-blocking RGD peptides (by 92+/-8%). The results suggest that the beta-adrenergic signaling pathway and integrin activation, which are known to interact, may play an important role in the response mechanism.     

Characterization of a pine multigene family containing elicitor- responsive stilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus. Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like UV-light or infection with the phytopathogenic fungus Botrytis cinerea. A genomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four different members of the five gene subclasses were characterized by sequencing. Emphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG. While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants and by transient expression in tobacco protoplasts. Constructs used contained the bacterial -glucuronidase under the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-specific expression in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinosylvin formation in the heartwood.     

Induction of pilocarpine formation in jaborandi leaves by salicylic acid and methyljasmonate

Jaborandi seedlings were subjected to different treatments in order to study the induction of pilocarpine in the leaves. In addition four extraction methods were assessed to extract the alkaloid from dried leaves. The highest yielding extraction and recovery was observed when dried leaves were first treated with base and then extracted with chloroform. Salt stress (NaCl), wounding, hypoxia, and N and K omission of the nutrient soln caused reductions in pilocarpine contents. Whereas complete nutrient soln and P omission maintained normal levels of the alkaloid. Salicylic acid and methyljasmonate induced a 4-fold increase of pilocarpine, but this increase was dependent on the concentration and time after exposure.     

Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D3-II: calcium transport, receptor kinetics, and signal transduction in intestine of female chickens

We recently reported (Larsson and Nemere [2003]: Endocrinology 144:1726) the effects of growth and maturation on 1,25(OH)2D3-membrane initiated effects in the intestine of male chickens. Here we extend our observations to studies on females with two stages of high calcium demand: growth (7-14 weeks) and egg laying (28-58 weeks). The rapid stimulatory effect of 130 pM 1,25(OH)2D3 on calcium transport was assessed as a physiological response in perfused duodena of 7-, 14-, 28-, and 58-week-old chickens, and determined to be 308%, 184%, 170%, and 153%, respectively, of corresponding controls after 40 min. Saturation analyses of [3H]1,25(OH)2D3 binding to nuclear vitamin D receptor (VDR) indicated an absence of cooperative binding, no changes in dissociation constant (Kd) with age, and an increase in maximum binding capacity (Bmax) between 7-week birds and older age groups. Analyses of saturable binding of [3H]1,25(OH)2D3 to the membrane associated rapid response steroid binding protein (1,25D3-MARRS bp) in basal lateral membranes (BLM), indicated cooperative binding, and an increase in both Bmax and Kd with age. No changes in the age-related expression of 1,25D3-MARRS bp were found, as judged by Western analyses, suggesting that a shift in ligand binding to lower affinity membrane components accounted for the increase in calculated Bmax. Basal levels of protein kinase C (PKC) activity decreased with age, as did hormone enhancement of activity. Basal levels of protein kinase A (PKA) activity remained constant with age, while the magnitude of hormone stimulation increased. Comparison of dose-response curves for ion transport and kinase activities in 7-week chicks suggested that PKC mediates phosphate transport while PKA mediates calcium transport. Thus, the age-related loss of calcium transport is most likely related to loss of PKC-mediated phosphate transport.