Evolution of photosensory pineal organs in new light: the fate of neuroendocrine photoreceptors

Pineal evolution is envisaged as a gradual transformation of pinealocytes (a gradual regression of pinealocyte sensory capacity within a particular cell line), the so-called sensory cell line of the pineal organ. In most non-mammals the pineal organ is a directly photosensory organ, while the pineal organ of mammals (epiphysis cerebri) is a non-sensory neuroendocrine organ under photoperiod control. The phylogenetic transformation of the pineal organ is reflected in the morphology and physiology of the main parenchymal cell type, the pinealocyte. In anamniotes, pinealocytes with retinal cone photoreceptor-like characteristics predominate, whereas in sauropsids so-called rudimentary photoreceptors predominate. These have well-developed secretory characteristics, and have been interpreted as intermediaries between the anamniote pineal photoreceptors and the mammalian non-sensory pinealocytes. We have re-examined the original studies on which the gradual transformation hypothesis of pineal evolution is based, and found that the evidence for this model of pineal evolution is ambiguous. In the light of recent advances in the understanding of neural development mechanisms, we propose a new hypothesis of pineal evolution, in which the old notion 'gradual regression within the sensory cell line' should be replaced with 'changes in fate restriction within the neural lineage of the pineal field'.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Influence of tidal volume on pulmonary NO release,tissue lipid peroxidation and surfactant phospholipids

Mechanical stress during ventilation may cause or aggravate acute lung injury. This study investigates the influence of low vs. high tidal volume (V(t)) on factors known to play key roles in acute lung injury: nitric oxide release, eNOS and iNOS gene expression, lipid peroxidation (LPO), and surfactant phospholipids (PL). Isolated rabbit lungs were subjected to one of three ventilation patterns for 135 min (V(t)-PEEP): 6 ml/kg-0 cm H(2)O. 12 ml/kg-0 cm H(2)O 6 ml/kg-5 cm H(2)O, 12 ml/kg-0 cm H(2)O, and 6 ml/kg-5 cm H(2)O resulted in comparable peak inspiratory pressure (PIP). This allowed comparing low and high V(t) without dependence on PIP. Ventilatory patterns did not induce changes in pulmonary artery pressure, vascular permeability (K(f,c)), PIP or pulmonary compliance. High V(t) in comparison with both of the low V(t) groups caused an increase in BALF-nitrite (30.6+/-3.0* vs. 21.4+/-2.2 and 16.2+/-3.3 microM), BALF-PL (1110+/-19* vs. 750+/-68 and 634+/-82 microg/ml), and tissue LPO product accumulation (0.62+/-0.051* vs. 0.48+/-0.052 and 0.43+/-0.031 nmol/mg), *P<0.05 each. Perfusate nitrite and BALF-PL composition (assessed by use of 31P-NMR spectroscopy and MALDI-TOF mass spectrometry) did not differ among the groups. High V(t) ventilation reduced eNOS gene expression but did not affect iNOS expression. The increased release of NO and the accumulation of LPO products may represent early lung injury while elevated BALF-PL may reflect distension-induced surfactant secretion.     

Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice

Genetic approaches have provided evidence that DNA end-joining problems serve an essential role in neuronal survival during development of mammalian embryos. In the present study, we tested whether the DNA repair enzyme, DNA dependent protein kinase, plays an important role in the survival of cerebral cortical neurons in mice. DNA-PK is comprised of a DNA-binding subunit called Ku and a catalytic subunit called DNA-PKcs. In mice with the scid mutation, DNA-PKcs is truncated near the kinase domain, which causes loss of kinase activity. We compared the spatial and temporal aspects of neuronal cell death in scid versus isogenic wild-type embryos and found a significant increase in dying cells in scid mice, as assessed by nuclear changes, DNA fragmentation and caspase-3 activity. Additional biochemical and immunocytochemical studies indicated that of several DNA repair enzymes investigated, only PARP was increased in scid mice, possibly in response to elevated DNA strand breaks.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2)

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

A selective somatostatin type-2 receptor agonist inhibits neointimal thickening and enhances endothelial cell growth and morphology following aortic balloon injury in the rabbit

Somatostatin analogs have been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and attenuate neointimal thickening following experimental balloon catheter injury. In this study, the effects of a selective agonist for the somatostatin receptor subtype 2, PRL-2486, on neointimal thickening and endothelial cell regrowth 2 weeks following balloon catheterization of male New Zealand White rabbits were determined. Rabbits treated 2 days prior to and 2 weeks after catheter injury with 10 g/kg/day PRL-2486 (PRL-tx) had decreased I/M ratios (intimal area/medial area × 100; p < 0.05) but had no effect at lower (5 g/kg/day) or higher (20 g/kg/day) doses. PRL-tx had significantly decreased VSMC proliferation compared to untreated animals. PRL-tx increased endothelial regrowth by over 2-fold (p < 0.002) and improved endothelial cell morphology. Endothelial-dependent relaxation responses to acetylcholine were attenuated by catheter injury, and were not improved with PRL-tx. These data suggest that the PRL-2486-mediated inhibition of neointimal thickening exhibits a bell-shaped dose-response curve. This inhibition may be due in part to decreased VSMC proliferation, which may be a function of enhanced endothelial regrowth, but not the return of endothelium-dependent vascular function.     

The gel-forming behaviour of dextran in the presence of KCl: a quantitative 13C and pulsed field gradient (PFG) NMR study

Although the gel forming ability of certain polysaccharides in the presence of ions is a well-known phenomenon, detailed physicochemical mechanisms of such processes are still unknown. In this investigation high resolution 13C NMR as well as 1H pulsed field gradient (PFG) NMR were used to investigate the mobility of dextran in the sol and in the gel state. Gel-formation of dextran can be easily induced by the addition of large amounts of potassium chloride. No major differences in the T(1) relaxation times of dextran in the sol and in the gel state could be observed. Accordingly, the analysis of the 13C NMR spectroscopic data did not provide any indication of an observable line-broadening upon gel-formation. However, a KCl concentration dependent decrease of signal intensity in comparison to an internal standard was detected. On the other hand, the PFG NMR studies clearly indicated a gradual diminution of the self-diffusion coefficient of the dextran with increasing molecular weight as well as in the presence of potassium chloride. These measurements revealed in agreement with spectroscopic data that at least one potassium ion per monomer subunit (i.e. one glycopyranose residue) is necessary for gel formation.