Detailed mapping of CGRP mRNA expression in the rat central nervous system: Comparison with previous immunocytochemical findings

The localization of CGRP mRNA in neurons of the rat brain and spinal cord was assessed by in situ hybridization histochemistry (ISH) using a radiolabeled synthetic 57-mer oiigodeoxynucleotide probe complementary to the rat prepro CGRP mRNA. Results were compared with previously published findings of CGRP-immunoreactive (CGRP-IR) cell bodies revealed by an indirect immunofluorescence technique. The highest numbers of CGRP mRNA expressing neurons as well as the greatest intensity of staining were found in the lateral hypothalamic area, the parabrachial nuclei, and among the cranial motor nuclei, especially in the nuclei of the 7th and 12th nerve and the ambiguus nucleus, which is generally in good agreement with findings assessed by immunocytochemistry (ICH). However, some mismatches between the localizaton of the peptide by ICH and the localization of the CGRP mRNA were also observed. Thus, ISH was not able to confirm CGRP-IR in cells of the amygdaloid complex and parts of the medial hypothalamus, the central gray, and the inferior colliculus, but ISH revealed considerably more CGRP mRNA expressing cells in the lateral hypothalamic area, arcuate nucleus, posterior and peripeduncular thalamic nuclei, and all cranial motor nuclei than CGRP-IR containing cells found by ICH. Moreover, ISH also revealed CGRP mRNA synthesis in the nucleus of the lateral olfactory tract and in the perihypoglossal nuclei that were devoid of CGRP-IR. The reasons for the observed mismatches still remain to be elucidated; however, intracerebroventricular colchicine pretreatment used to increase immunocytochemical signals also might have induced or suppressed gene expression in certain brain regions in an unpredictable matter. On the other hand, detection of only the mRNA in a certain region does not necessarily mean that also the active peptide is synthesized there.     

Catabolism of newly synthesized decorin in vitro by human peritoneal mesothelial cells.

OBJECTIVE: Previous studies have shown that decorin and biglycan account for over 70% of the proteoglycans (PGs) synthesized by human peritoneal mesothelial cells (HPMCs). Since these PGs are involved in the control of cell growth, cell differentiation, and matrix assembly, we investigated their turnover in cultured HPMCs. METHODS: Confluent HPMCs were metabolically labeled with [35S]-sulfate and the labeled products isolated from the cell medium and the cell layer characterized by sensitivity to bacterial eliminases. Experiments were undertaken with exogenous labeled decorin, and its metabolic state was studied. RESULTS: In a 24-hour labeling period, 75% of the newly synthesized chondroitin sulfate/dermatan sulfate (CS/DS) PGs appeared in the culture medium, the majority of which (90%) was decorin. In the cell layer, protein-free glycosaminoglycan (GAG) chains accounted for 21% of the total CS/DS at 24 hours and exhibited constant specific activity at 12-16 hours. The latter material was turned over with a half-life of approximately 2.5 hours. Exogenous decorin underwent receptor-mediated endocytosis and subsequent intracellular degradation. Uptake but not degradation could be inhibited by heparin. CONCLUSIONS: HPMCs are distinguished by a rapid turnover of decorin. A characteristic metabolic feature is the existence of a large intracellular pool of protein-free DS-GAGs. Understanding the control of decorin turnover in HPMCs might lead to delineation of its potential role in both the physiology and pathophysiology of the membrane in PD patients.     

Influence of continuous infusion of interleukin-1 alpha on the core protein and the core protein fragments of the small proteoglycan decorin in cartilage.

Decorin, a collagen-binding small proteoglycan, is considered to have a specific function in the organization or stability of the collagen network. Therefore, alteration of its molecular properties may be of pathophysiological relevance during the development of cartilage damage. It is shown here that normal cartilage from rabbit knee-joint contains glycosaminoglycan chain-bearing core protein fragments of 39, 23, and 18 kDa, each one amounting to approximately 5-6% of the intact decorin core protein. Continuous infusion of human recombinant interleukin-1 alpha for 14 days (200 ng/day) into a knee-joint led in condylar cartilage to a reduction in the amount of intact core protein from 2 micrograms/mg wet tissue to about 1.1 micrograms/mg. The increase in its quantity found after infusion of heat-inactivated interleukin-1 was not statistically significant. The concentration of all three core protein fragments became reduced to a similar extent as the intact core protein under the influence of the cytokine, and additional fragments were not found. Surprisingly, there was a much smaller response to interleukin-1-treatment in patellar cartilage.     

Influence of renal replacement therapy on outcome of patients with acute renal failure

There are many controversial results about the influence of acute renal failure (ARF) and renal replacement therapy (RRT) on patient outcome in intensive care units. This retrospective study compared demographics. severity, course, and prognosis of ARF during 36 months (period 1, 1991 through 1993; 128 cases) and 18 months (period 2, 1994 through 1995; 141 cases). Compared with period 1, during period 2 there was a markedly increased incidence of ARF. There were no significant differences in patient demographics or etiology of renal failure, but the therapeutic approach to ARF was quite different. During period 2, RRT was started at earlier stages of renal insufficiency (that is, less elevated creatinine serum concentrations or reduced diuresis). Additionally, there was a significant increase in the numbers of continuous RRT (CRRT) replacing the discontinuous mode of dialysis treatment. Compared with period 1, mortality was reduced from 78.9 to 59.6% during period 2 (P < 0.001). There were no differences in mortality between the patients from internal and surgical wards. Mortality in patients treated with CRRT was in period 1 and in period 2 higher than mortality in patients treated with intermittent RRT, but these results are biased by a preferred use of CRRT in severely ill patients with an unstable circulatory system. These data suggest that the early onset of RRT reduces the mortality of intensive care unit patients with ARF independent of underlying diseases. An influence of the method of RRT, sex, and age on outcome of patients with ARF could not be proven.     

Concentration gradients in evaporating binary droplets probed by spatially resolved Raman and NMR spectroscopy

Understanding the evaporation process of binary sessile droplets is essential for optimizing various technical processes, such as inkjet printing or heat transfer. Liquid mixtures whose evaporation and wetting properties may differ significantly from those of pure liquids are particularly interesting. Concentration gradients may occur in these binary droplets. The challenge is to measure concentration gradients without affecting the evaporation process. Here, spectroscopic methods with spatial resolution can discriminate between the components of a liquid mixture. We show that confocal Raman microscopy and spatially resolved NMR spectroscopy can be used as complementary methods to measure concentration gradients in evaporating 1-butanol/1-hexanol droplets on a hydrophobic surface. Deuterating one of the liquids allows analysis of the local composition through the comparison of the intensities of the C–H and C–D stretching bands in Raman spectra. Thus, a concentration gradient in the evaporating droplet was established. Spatially resolved NMR spectroscopy revealed the composition at different positions of a droplet evaporating in the NMR tube, an environment in which air exchange is less pronounced. While not being perfectly comparable, both methods—confocal Raman and spatially resolved NMR experiments—show the presence of a vertical concentration gradient as 1-butanol/1-hexanol droplets evaporate.

Evaporating droplets occur in various contexts such as inkjet printing (1, 2), heat transfer, or daily phenomena such as drying coffee stains (3, 4). In many applications, such as painting (5), cleaning, gluing, or printing (6), where liquid mixtures are used, the evaporation of a droplet is a complex process because the concentration profile within the droplet varies over time. To improve the controllability and predictability of the technical processes, it is essential to characterize the transport phenomena during the drying process. The measurement of the droplet composition is a crucial element and has to be carried out with sufficient spatial and temporal resolution. In particular, spectroscopic methods are promising tools for contactless concentration measurements of liquid mixtures.The evaporation of a droplet is governed by physical properties such as surface tension (7), density (8–10), vapor pressure (11), and boiling temperature. Additionally, concentration gradients can evolve in liquid mixtures (12). These gradients are driven by thermal gradients due to the enthalpy of evaporation (droplet cooling) or on heated surfaces, by surface tension gradients induced by preferential evaporation of one component or by density gradients for droplets composed of liquids with different densities like water and glycerol (13). The evaporation rates of the components can vary over the droplet surface. For sessile droplets with contact angles smaller than 90°, for example, the evaporation rates are higher at the three-phase contact line (14). These thermal or surface tension gradients can induce flow inside the droplet called Marangoni flow. This flow leads to concentration gradients across the droplet (7–10). The direction of the gradient depends on the density and surface tension. A direct application of this principle is, for instance, Marangoni cleaning in semiconductor technology (15).The investigation of the composition of sessile drops on the microliter scale, as they occur in inkjet printing or other technical processes, poses a challenge because the typical length scales of interest are smaller than the capillary length. In bulk samples, the composition can be examined in a straightforward manner with chromatographic methods such as gas chromatography and high-performance liquid chromatography or spectroscopic methods such as NMR spectroscopy, infrared spectroscopy, and Raman spectroscopy. However, for the investigation of sessile droplets, a high spatial and temporal resolution is required. For this purpose, confocal Raman spectroscopy and spatially resolved NMR spectroscopy are powerful tools. For both techniques, concentration determination is straightforward if at least two signals of the components of interest are baseline-separated. NMR is intrinsically calibration-free, whereas Raman spectroscopy requires calibration through reference experiments (16–18). Both approaches allow the quantification of concentration gradients in sessile droplets, as is shown here.In Raman microscopy, good spatial resolution can be achieved in a confocal setup. The components of mixtures can be distinguished via specific vibrations for different functional groups or through a careful analysis of the Raman signals in the fingerprint region (<1,500 cm⁻¹). For example, binary mixtures of ethanol and water can be characterized in a straightforward manner (17). If, however, both liquids have a similar chemical structure, the discrimination of the components might be hampered by signal overlap in the C–H stretching region (2,800 to 3,000 cm⁻¹); e.g., in such cases, Raman signals in the fingerprint region (<1,500 cm⁻¹) might be used for the identification of the species. However, these signals often provide a poor signal-to-noise ratio, which makes large integration times necessary. Thus, the image rate or resolution is so low that even slow diffusion processes are hardly resolved. Here, Raman stable isotope probing (SIP), which has been developed to monitor metabolic processes in microbiology, offers a solution (19). The basic idea of Raman SIP is to replace the proton in the C–H with deuterium in one of the mixture components such that the C–D stretching region occurs at roughly 1/2 times the C–H stretching and falls into a region with very weak or even without signals from the protonated liquid component. Thus, the concentration in a binary mixture can be calculated in a straightforward manner from the ratio of the integrated Raman intensities ICD/ICH of the respective stretching vibrations.Compared to Raman microscopy, where localization is achieved by scanning the focal point across the region of interest, in NMR experiments localization is achieved by using magnetic field gradients. Usually, one avoids phase boundaries (especially liquid–gas interfaces) in NMR experiments because they disturb the magnetic field homogeneity and reduce the spectral quality in terms of line shape and baseline separation of the resonances. Nevertheless, it has been shown that MRI can be used to characterize freezing water droplets (20), the infiltration of water into asphalts (21), and the evaporation of sessile droplets from porous surfaces (22–24). Additionally, NMR can be used to quantify the composition of binary droplets during evaporation (25).Thus, the use of both complementary approaches to characterize evaporating binary droplets may be beneficial. In this article, we discuss the capabilities of Raman SIP and NMR techniques to analyze the evolution of the composition of an evaporating sessile binary droplet. As a model system, a binary mixture of 1-butanol and 1-hexanol was used. This mixture shows a low volatility such that the evaporation process can be captured with both Raman and NMR spectroscopies. With Raman spectroscopy, it was possible to observe concentration gradients of 1-butan-d₉-ol over the height of the droplet during evaporation. NMR techniques were examined in terms of the capability to observe the evaporation of 1-butanol and yield time-dependent droplet composition with spatially resolved ¹H-NMR spectra. Furthermore, the contours of the evaporating droplets were tracked by optical measurements to characterize the time-dependent changes in the droplet dimensions. Flows induced by the concentration gradients were confirmed by astigmatic particle tracking velocimetry.     

Language and Aggressive Behaviors in Male and Female Youth with Autism Spectrum Disorder

Journal of Autism and Developmental Disorders - Aggressive behaviors are common among youth with autism spectrum disorder (ASD) and correlate with pervasive social-emotional difficulties....     

Deficient expression of the small proteoglycan decorin in a case of severe/lethal osteogenesis imperfecta

In osteogenesis imperfecta (OI) the effects of mutations in type I collagen genes generally reflect their nature and localization. Unrelated individuals sharing identical mutations present, in general, similar clinical phenotypes. However, in some such cases the clinical phenotype differs. This variable clinical expression could be the result of abnormalities in other connective tissue proteins. Since decorin is a component of connective tissue, binds to type I collagen fibrils and plays a role in matrix assembly, we studied decorin production in skin fibroblasts from OI patients. Cultured fibroblasts from one patient with extremely severe osteogenesis imperfecta (classified as type II/III) who has an α1(I)gly415ser mutation were found to secrete barely detectable amounts of decorin into culture medium. Western blotting using antibodies raised against decorin confirmed the reduction of the decorin core protein and Northern blot analysis showed decorin mRNA levels below the limit of detection. Cells from a patient, with a less severe phenotype, bearing a mutation in the same position of the triple helix (α1(I)gly415) expressed decorin normally. The different clinical phenotypes could be due to the differing genetic backgrounds of the patients so it is tempting to conclude that in our most severely affected patient the absence of decorin aggravates the clinical phenotype. © 1996 Wiley-Liss, Inc.     

Clinical heterogeneity in Sanfilippo disease (mucopolysaccharidosis III) type D: presentation of two new cases

Clinical, radiological and biochemical findings of two new cases of Sanfilippo disease, type D are reported. A high percentage of heparan sulfate was found in the urinary glycosaminoglycan pattern and a severe deficiency of N-acetylglucosamine-6-sulfate sulfatase was demonstrated in skin cultured fibroblasts from the patients. One of the patients presented mild intellectual impairment which differentiates him from the other cases described to date.