Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway.

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-kappaB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Specific function of B-Raf in mediating survival of embryonic motoneurons and sensory neurons.

Embryonic sensory and motoneurons depend on neurotrophic factors for survival. Here we show that their survival requires B-Raf, which, in this function, cannot be substituted by C-Raf. Sensory and motoneurons from b-raf-deficient mice do not respond to neurotrophic factors for their survival. However, these primary neurons can be rescued by transfection of a b-raf expression plasmid. In contrast, c-raf-deficient neurons survive in response to neurotrophic factors, similarly to neurons from wild-type mice. This points to an essential and specific function of B-Raf in mediating survival of sensory and motoneurons during development.

The anti-apoptotic protein ITA is essential for NGF-mediated survival of embryonic chick neurons.

The avian ITA is homologous to the baculoviral and mammalian inhibitor of apoptosis (IAP) proteins, which can prevent apoptosis by inhibition of specific caspases. We investigated the role of ITA in embryonic chick sympathetic and dorsal root ganglionic neurons, which depend on nerve growth factor (NGF) for their survival. Within 6 hours, NGF upregulated ITA protein production more than 25-fold in sensory and sympathetic neurons. Overexpression of ITA in primary neurons supported survival of these cells in the absence of NGF, and ita antisense constructs inhibited NGF-mediated survival. Thus the induction of ITA expression seems to be an essential signaling event for survival of sympathetic and dorsal root ganglionic sensory neurons in response to NGF.

Advances in the treatment of portal hypertension in cirrhosis.

Non-selective beta-blockers and handling of esophageal varices has been key elements in the treatment of portal hypertension in recent decades. Liver vein catheterization has been essential in diagnosis and monitoring of portal hypertension, but ongoing needs for noninvasive tools has led to research in areas of both biomarkers, and transient elastography, which displays promising results in discerning clinically significant portal hypertension. Novel research into the areas of hepatic stellate cell function and the dynamic components of portal hypertension has revealed promising areas of treatment modalities, targeting intestinal decontamination, angiogenesis, inflammation and oxidative stress. Future studies may reveal if these initiatives lead to developments of new drugs for treatment of portal hypertension.

Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons.

Developing motoneurons require trophic support from their target, the skeletal muscle. Despite a large number of neurotrophic molecules with survival-promoting activity for isolated embryonic motoneurons, those factors that are required for motoneuron survival during development are still not known. Cytokines of the ciliary neurotrophic factor (CNTF)-leukemia inhibitory factor (LIF) family have been shown to play a role in motoneuron (MN) survival. Importantly, in mice lacking the LIFRbeta or the CNTFRalpha there is a significant loss of MNs during embryonic development. Because genetic deletion of either (or both) CNTF or LIF fails, by contrast, to perturb MN survival before birth, it was concluded that another ligand exists that is functionally inactivated in the receptor deleted mice, resulting in MN loss during development. One possible candidate for this ligand is the CNTF-LIF family member cardiotrophin-1 (CT-1). CT-1 is highly expressed in embryonic skeletal muscle, secreted by myotubes, and promotes the survival of cultured embryonic mouse and rat MNs. Here we show that ct-1 deficiency causes increased motoneuron cell death in spinal cord and brainstem nuclei of mice during a period between embryonic day 14 and the first postnatal week. Interestingly, no further loss was detectable during the subsequent postnatal period, and nerve lesion in young adult ct-1-deficient mice did not result in significant additional loss of motoneurons, as had been previously observed in mice lacking both CNTF and LIF. CT-1 is the first bona fide muscle-derived neurotrophic factor to be identified that is required for the survival of subgroups of developing motoneurons.

The genes for human brain factor 1 and 2, members of the fork head gene family, are clustered on chromosome 14q.

Brain factor-1 (BF-1) is a member of the fork head gene family which shows expression restricted to the neurons of the developing telencephalon in rodents and man. We have isolated a second human gene (HBF-2), which is also strongly expressed in embryonic brain and has very high homology to both the rat and human brain factor-1 genes and the retroviral oncogene qin. The HBF-2 cDNA was isolated from a human fetal brain expression library and contains a putative open reading frame of 479 amino acids. The HBF-2 gene is strongly expressed in fetal brain and also with lower levels of expression in several adult tissues. At the genomic level the gene for HBF-1 contains an 500 bp intron situated between the DNA binding domain II and the fork head domain while that of HBF-2 is intronless. The two genes are clustered on human chromosome 14q11-13.

Gene expression of brain natriuretic peptide in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length.

BACKGROUND: We studied the effects of angiotensin II (Ang II) and diastolic overstretch on the induction of cardiac growth in isometrically contracting muscle preparations from human right atria and left ventricles. We used the gene expression of brain natriuretic peptide (BNP) as a molecular marker of cardiac hypertrophy. METHODS AND RESULTS: Northern blot analysis was performed in human atrial muscle preparations, which were either incubated in 10(-6) mol/L Ang II for 45 minutes or diastolically stretched to 120% of optimum muscle length. Similar experiments were performed with human left ventricular muscle preparations. Results were as follows: (1) BNP gene expression increased in human atrial myocardium 4-fold when stimulated by Ang II (n=7, P<0.001). (2) Diastolic overstretch increased BNP expression in a time-dependent manner. The linear regression equations for the BNP/GAPDH ratio as a function of time (hours) were y=1.21+0.62x (P:<0.001) for overstretched preparations and y=1.07-0.01x (P:=NS) for atrial preparations kept at physiological muscle length. (3) In left ventricular human muscle preparations, diastolic overstretch and Ang II increased BNP gene expression as well. (4) In addition, the Ang II subtype 1 receptor blocker losartan was able to block the effects of Ang II and diastolic overstretch. CONCLUSIONS: Cardiac hypertrophy can be induced in isolated human atrial and left ventricular intact myocardium by Ang II and diastolic overstretch but not by isometric afterload. The fact that the induction of cardiac growth is inhibited by the blockade of Ang II subtype 1 receptors is of scientific and clinical importance.

Isolation and culture of spinal cord motor neurons.

Isolated spinal motoneurons are a powerful tool for studying basic mechanisms of neurite growth and survival. Since motoneurons are a minor population of developing spinal cord cells, they need to be purified and enriched to separate them from non-neuronal cells. Therefore, the particular feature of embryonic motoneurons to express the low affinity neurotrophin receptor p75(NTR) is used to separate the motoneurons from other contaminating cells. Two ways are described to isolate embryonic motoneurons: the basic protocol taking advantage of the ability of p75(NTR) to bind lectin, and an alternative method using an antibody against p75(NTR) for a panning procedure. These protocols comprise suggestions for the cultivation of the isolated motoneurons for experiments regarding neural outgrowth and survival as well as instruction for the preparation of proteins of the cells.

Nitric oxide synthase in the brain of the turtle Pseudemys scripta elegans.

The distribution pattern of nitric oxide synthase (NOS) was investigated in the brain of the turtle by NADPH-diaphorase histochemistry. The specificity of the histochemical staining was tested by immunocytochemical colocalization with an antiserum specific for NOS. In the forebrain, neurons staining intensely for nitric oxide synthase were localized in the olfactory tubercle, the basal ganglia complex, the basal amygdaloid nucleus, suprapeduncular nucleus, and the posterior hypothalamic area. Many positive fibers course in a tract connecting the basal amygdaloid nucleus with the hypothalamus, corresponding to the stria terminalis. Bundles of nitroxergic fibers were seen to course at the ventromedial edge of the optic tract and to cross in the supraoptic decussation, apparently consisting of tectothalamic and thalamotectal fibers. In the midbrain, strongly NOS-positive neurons were present in the substantia nigra, the nucleus profundus mesencephali, the periventricular grey of the optic tectum, the laminar nucleus of the torus semicircularis, and the nucleus of the lateral lemniscus. The area of the locus coeruleus harbored an accumulation of intensely stained neurons, which, as in mammals, might represent a cholinergic cell group of the reptilian brainstem. In the cerebellum, strong staining was confined to bundles of afferent fibers running in the lower molecular and in the Purkinje cell layer. These axons appeared to include ascending projections from the dorsal funicular nucleus or the spinal cord. NOS-positive cells in the caudal brainstem were found in the cerebellar nuclei, in the superior vestibular nucleus, in the reticular nuclei, ventrolateral to the nucleus of the solitary tract, in the perihypoglossal, and in the dorsal funicular nucleus. Taken together, these results suggest that nitric oxide acts as a messenger molecule in different areas of the reptilian brain and spinal cord. In certain areas, the pattern of expression of NOS appears to have evolved before radiation of present mammalian, avian, and reptilian species.

The novel human HNF-3/fork head-like 5 gene: chromosomal localization and expression pattern.

Analysis of cDNA clones, isolated from a human fetal brain cDNA library, that hybridized with the rat HNF-3 alpha fork head homolog domain revealed the 3.6-kb HFKL5 cDNA. The transcript of HFKL5 is 4.4 kb long and represents a novel member of the HNF-3/fork head transcription factor family. Comparison of the amino acid sequence of the fork head domain reveals a relatively low level of homology to other members of this family of genes, the closest related sequence being rat HFH7 with 68% homology. The HFKL5 cDNA codes for a putative 500-amino-acid protein. Southern analysis revealed that the HFKL5 gene homolog is present as a single copy in the human genome. Zoo Southern analysis showed strong evolutionary conservation of HFKL5 among mammalian and possibly avian species. Expression of HFKL5 in neurons is restricted to the fully differentiated neurons in fetal and adult brain as well as in the parasympathic ganglia of the small intestine. We also observed expression in lymphocytes, kidney tubule cells, and a subset of hepatocytes. The HFKL5 gene homolog was mapped to chromosome 22q13-qter by cell panel hybridization.

Effect of substrate manipulation on reducing ischemia/reperfusion injury in isolated perfused rat hearts.

The objective of this investigation was to assess the effect of substrate manipulation on reducing ischemia/reperfusion injury (IRI). Isolated rat hearts were perfused with modified Krebs-Henseleit buffer containing either (in mM): glucose 11 (G1), glucose 22 (G2), or glucose 11 with either xylitol 11 (GX), mannitol 11 (GM), L-leucine 1 (GL), or L-glutamic acid 2 (GGA), respectively. Hearts were subjected to 10 min of global no-flow ischemia, followed by 20 min of reperfusion. Mean tissue perfusion, oxygen consumption, and peak left ventricular pressure (PLVP) were determined at baseline, in the first minute of regular heart rhythm following ischemia, and after 20 minutes of reperfusion. Reperfusion arrhythmia (in sec) was significantly (all p < 0.05) shorter in GGA (115 +/- 33) vs G1 (315 +/- 29) and G2 (273 +/- 33), and also in GL (161 +/- 26) vs G1. Dry/wet heart weight ratios were also greater in GGA (0.20), when compared with G2 (0.16), GX (0.17), GM (0.17), GM (0.17), and GL (0.17) (all p < 0.02), suggesting less cellular/interstitial edema. Percent recovery in PLVP was improved (p < 0.03) in GL (81 +/- 2) and GGA (81 +/- 2) vs. G2 (71 +/- 3), without significant alterations in oxygen consumption. Thus, cardiac IRI can be diminished by substrate manipulation, especially by augmentation of glutamate and leucine, most likely due to an improved anaerobic energy generation and utilization.

Impact of specific substrate supply on efficiency of cardiac function: an update.

Investigations of the mechanisms that modulate energy generation during states of altered cardiac metabolism have reached a point where there is both need and demand for novel approaches. The evidence discussed here strongly suggests that both energy generation and utilization in these states may be effectively strengthened by nutritional manipulation. Compared with standard treatments for ischemia/reperfusion injury or heart failure, nutritional therapy may present an important and less toxic approach by affecting the mechanisms of energy utilization during compromised cardiac states. We provide not only a conceptual framework for further experimental studies of myocardial metabolism during ischemia and reperfusion injury but also a basis for developing clinically applicable nutrients designed to improve deranged cardiac function. The use of traditional energy substrates, in conjunction with those that may be conditionally important during compromised cardiac states, potentially offers a useful therapeutic modality in the treatment of the cardiac patient.

Branched-chain amino acids and respiration.

A series of investigations suggest a specific role for BCAA in the regulation of respiration. In vitro incubation studies have shown that BCAAs improve the recovery of muscle force after fatigue. Further investigations revealed that leucine plays a key role in this action and acts in a manner not dependent on its use as an energy substrate. In humans, solutions enriched with BCAA have decreased PCO2 and stimulated the ventilatory response to hypercapnia, thereby corresponding to an enhanced ventilatory sensitivity with the administration of BCAA. The mechanisms for these actions are unknown. The most viable hypothesis is based on the ability of BCAA to decrease the synthesis of serotonin due to altered transport of AAs, including tryptophan, to the brain. Clinical studies have suggested a potency of BCAA in the treatment of respiratory dysfunction of preterm infants, as well as of patients with sleep apnea related to various disease states. The clinical applications of BCAA-enriched mixtures in respiratory diseases are still experimental, and many controversies exist concerning the validity of BCAA in clinical practice. Most TPN regimens contain BCAA approximating the average intake of BCAA in the Western diet. The question therefore remains whether additional BCAA supplementation is useful to achieve the suggested metabolic and pharmacological effects. Meticulous future studies are needed to establish the therapeutic value of BCAA in the treatment of various respiratory functions.

Signalling mechanisms for survival of lesioned motoneurons.

Mechanisms controlling neuronal survival play an important role both during development and after birth, in particular when the nervous system is lesioned. Isolated embryonic motoneurons and other types of primary neurons have been a useful tool for studying basic mechanisms underlying neuronal cell death during development and under pathophysiological conditions after neurotrauma. These studies have led to the identification of neurotrophic factors which under physiological conditions regulate survival and functional properties, and after neurotrauma promote regeneration and plasticity. Functional analysis of these molecules, in particular by generation of gene knockout mice, has led to a more detailed understanding of complex requirements of individual types of neurons for their survival and also paved the way for a better understanding of the signalling pathways in lesioned neurons which decide on cell death or survival after axotomy and other pathophysiological conditions. These findings could ultimately lead to a rational basis for therapeutic approaches aiming at improving neuronal survival and regeneration after neurotrauma.

Mechanical and excitotoxic lesion of motoneurons: effects of neurotrophins and ciliary neurotrophic factor on survival and regeneration.

Mechanical lesion of peripheral nerves leads to extensive death of corresponding motoneurons in newborn rodents. The extent of cell death can be significantly reduced by neurotrophic factors. These molecules are produced by glial and neuronal cells and play an important role in supporting survival and regeneration of various neuronal populations in the central nervous system, in particular after mechanical, excitotoxic and ischemic insults. In addition, factors such as ciliary neurotrophic factor and neurotrophin-3 influence glial cell proliferation and survival. We have investigated the role of neurotrophic factors on motoneurons, both in cell culture and after axotomy in vivo. Moreover, the role of excitatory neurotransmission in modulating dendritic architecture of these cells was analyzed. Our data suggest that motoneurons are a suitable model for investigating the complex functional and morphological changes after brain lesion and for the identification of new therapeutic strategies to influence survival and functional recovery under such circumstances.

Three-dimensional nickel ion transport through porous media using magnetic resonance imaging.

The transport of Ni2+ ions in a column, filled with porous media, was observed in three dimensions and time by magnetic resonance imaging (MRI) in a clinical scanner. For porous media we used glass beads or quartz sand in a saturated continuous flow mode. The magnetic moment of Ni2+ decreased the T1 relaxation time of 1H in aqueous solution. This concentration-dependent effect was used by a fast low angle shot (FLASH) MRI sequence for imaging the concentration of the dissolved ions. Since Ni2+ behaves as a conservative tracer under the chosen conditions, the tracer motion was representative for the water flow in the porous medium. Currently, we can achieve an isotropic spatial resolution of 1.5 mm and a temporal resolution of 170 s. The transport observation gives direct access to hydraulic flow properties of the porous media. The fluid flow velocity field was calculated by a fronttracking method and the statistical properties of the velocities were investigated. We also compared the experimental data with the three-dimensional particle tracking model PARTRACE, which uses the experimental flow field as input.

[Results of pars plana lensectomy for childhood cataract]. / Ergebnisse der Pars-plana-Lentektomie bei kindlicher Katarakt.

BACKGROUND: Pars plana lensectomy with subsequent fitting of contact lenses is the standard procedure for cataracts occurring within the first 2 years of life. We wanted to assess the outcome and complication rate of this procedure. METHODS: Pars plana lensectomy was performed on 29 eyes of 15 children with bilateral as well as 12 eyes with unilateral cataracts. All children were reexamined at an age of at least 3 years. RESULTS: In bilateral cases the mean age at the time of surgery was 4.0 months and mean resulting visual acuity 0.32. After exclusion of three eyes with complications or bad postoperative compliance, we found a statistically significant correlation between age at surgery and visual acuity (r(2)=0.432, p<0.05). Some form of binocular vision was achieved by 40% of the children; 17% developed ocular hypertension and 7% a secondary cataract. In the unilateral cases the mean age at surgery was 3.9 months and the mean resulting visual acuity 0.14. There was no significant correlation between age and visual acuity; 17% gained binocular function and 5% had ocular hypertension. CONCLUSION: Visual function after lensectomy is better in eyes with bilateral cataracts compared to unilateral cataracts. Early surgery as well as adequate orthoptic therapy and compliance with wearing the contact lens are necessary for good outcome.

Spectrophotometric assessment of the effectiveness of Opalescence PF 10%: a 14-month clinical study.

OBJECTIVES: To evaluate the effectiveness of Opalescence PF 10% just after treatment, at 6-month follow-up and at a 14-month follow-up period. METHODS: Opalescence PF 10% was applied nightly for 14 days. The color of teeth 11 and 21 of 17 subjects were measured with a spectrophotometer (L*; a*; b*) before treatment, just after treatment (14 days), after 6 months and after 14 months. Subjects were instructed to take note of any tooth sensitivity. RESULTS: For all three components (L*, a* and b*) statistical significant differences (p<0.05) in the values between base-line, after treatment (14 days later), after 6 months and after 14 months were found (Wilcoxon Signed Rank Sum Test). The decrease in L* was about 20% after 6 months and about 50% after 14 months. The a* value decreased approximately 14% after 6 months but was worse after 14 months than at the beginning. The b* value decreased the least with about 9% after 6 months and about 8% after 14 months. The decrease in DeltaE(ab)(*) was approximately 20% after 14 months. Less than 20% of the subjects experienced mild tooth sensitivity just after treatment. CONCLUSION: Significantly whiter teeth were found after treatment as well as after a 6-month follow-up period. The whiteness/brightness (L*) decreased with approximately 50% after 14 months and the a* value with approximately 50% after 7 months, while the yellowness (b* value) remained even after 14 months. CLINICAL IMPLICATIONS: The product is an effective tooth whitener resulting in only low tooth sensitivity. Re-bleaching could be done at about 14 months.

High-temperature liquid chromatography. Part II: Determination of the viscosities of binary solvent mixtures--implications for liquid chromatographic separations.

This paper is the second in a series of consecutive publications, explaining the concept of high temperature liquid chromatography under various important aspects. The second publication deals with the determination of the viscosity of binary solvent mixtures used in reversed phase liquid chromatography in a temperature range between 25 and 250 degrees C. In literature, only limited data of the temperature dependent viscosities of liquid solvents or binary solvent mixtures can be found. Therefore, the viscosities of the pure solvents as well as the binary mixtures had to be determined experimentally up to 250 degrees C. The viscosity data were used to estimate the pressure drop in a capillary connecting a high-temperature HPLC system with a mass spectrometer. The solvent perturbation could be avoided by adjusting the diameter of the transfer capillary to the viscosity and vapour pressure of the mobile phase. The viscosity data were also used to show that a significant gain in analysis speed is theoretically feasible. This factor clearly depends on the nature of the solvent system, because for mixtures with a large viscosity maximum at ambient temperature, this effect is most pronounced.