Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO₄. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.     

Reactivation in Working Memory: An Attractor Network Model of Free Recall

The dynamic nature of human working memory, the general-purpose system for processing continuous input, while keeping no longer externally available information active in the background, is well captured in immediate free recall of supraspan word-lists. Free recall tasks produce several benchmark memory phenomena, like the U-shaped serial position curve, reflecting enhanced memory for early and late list items. To account for empirical data, including primacy and recency as well as contiguity effects, we propose here a neurobiologically based neural network model that unifies short- and long-term forms of memory and challenges both the standard view of working memory as persistent activity and dual-store accounts of free recall. Rapidly expressed and volatile synaptic plasticity, modulated intrinsic excitability, and spike-frequency adaptation are suggested as key cellular mechanisms underlying working memory encoding, reactivation and recall. Recent findings on the synaptic and molecular mechanisms behind early LTP and on spiking activity during delayed-match-to-sample tasks support this view.     

Histone Deacetylase Inhibition Enhances Tissue Plasminogen Activator Release Capacity in Atherosclerotic Man

The expression of the tissue plasminogen activator (t-PA) gene appears to be under epigenetic control and can be affected by histone deacetylation inhibition. The study aimed to test if histone deacetalyase inhibitor treatment lead to increased t-PA release or reduced exhaustion in t-PA release in response to stimulation, as well as change in plasminogen activator inhibitor-1 (PAI-1) in subjects with coronary disease. In this clinical study, 16 post-myocardial infarction subjects, the perfused forearm model was used with isoprenaline provocation during 20 minutes, to stimulate local t-PA release. Each subject was measured twice on the same day (repeated stimuli sequences) as well as on two different occasions, without treatment and after four weeks of treatment with valproic acid (500 mg, twice daily). Net forearm release for t-PA in response to isoprenaline at minutes 1.5, 3, 6, 9, 12, 15 and 18 was measured, allowing assessment of cumulative t-PA release. There was a reduction in the exhaustion of cumulative t-PA release during repeated and prolonged stimulation with valproic acid treatment compared to non-treatment. Plasma PAI-1 antigen was decreased following treatment compared to non-treatment (18.4 ± 10.0 vs. 11.0 ± 7.1 nanograms/ml respectively, mean with 95% confidence interval). These findings demonstrate that histone deacetylation inhibition increases the capacity for endogenous t-PA release in subjects with vascular disease. Furthermore, the fibrinolytic balance is favored with suppressed PAI-1 levels. More studies are needed to establish the clinical relevance of these findings.

Trial registration

EU Clinical Trials Register 2012-004950-27     

Regional Risk Assessment of a Brazilian Rain Forest Reserve

The objective of this study was to identify subareas inside and near an Atlantic Rain Forest reserve, the Parque Estadual Turístico do Alto Ribeira (PETAR), most likely to be affected by land use in the vicinity of the area. In addition, the study aimed to compare risks per stressor source (agriculture, human settlements and mining) to both epigean (surface) and hypogean (subterranean) aquatic fauna. The methodological approach included the relative vulnerability of endpoints to the stressors (pesticides, metals, nutrients, and particles) and ranking of stressor sources and habitats (epigean and hypogean streams) based on their relative distribution in 14 subareas within the catchment areas of the main rivers that cross PETAR: Pilões, Betari and Iporanga. Four subareas presented high risk for both epigean and hypogean fauna. Three of those areas were located inside the Betari catchment area, where most of the settlements and abandoned lead mines are located. The fourth area was situated in the headwaters of the Pilões River, where agricultural activities are intense. Agriculture and human settlements were the activities most likely to cause impacts on aquatic ecosystems. Uses of risk assessment results include management of the PETAR and communication to stakeholders by the Park Administration.     

DegePrime,a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies

The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571–1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.     

An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)₂ vitamin D₃ significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.