Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin

Here, we show a role for the RB1 family proteins in directing full heterochromatin formation. Mouse embryonic fibroblasts that are triply deficient for RB1 (retinoblastoma 1), RBL1 (retinoblastoma-like 1) and RBL2 (retinoblastoma-like 2) - known as TKO cells - show a marked genomic instability, which is coincidental with decreased DNA methylation, increased acetylation of histone H3 and decreased tri-methylation of histone H4 at lysine 20 (H4K20). Chromatin immunoprecipitation showed that H4K20 tri-methylation was specifically decreased at pericentric and telomeric chromatin. These defects are independent of E2F family function. Indeed, we show a direct interaction between the RB1 proteins and the H4K20 tri-methylating enzymes Suv4-20h1 and Suv4-20h2, indicating that the RB1 family has a role in controlling H4K20 tri-methylation by these histone methyltransferases. These observations indicate that the RB1 family is involved in maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin, linking tumour suppression and the epigenetic definition of chromatin.     

Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast

In contrast to animal and fungal cells, green plant cells contain one or multiple chloroplasts, the organelle(s) in which photosynthetic reactions take place. Chloroplasts are believed to have originated from an endosymbiotic event and contain DNA that codes for some of their proteins. Most chloroplast proteins are encoded by the nuclear genome and imported with the help of sorting signals that are intrinsic parts of the polypeptides. Here, we show that a chloroplast-located protein in higher plants takes an alternative route through the secretory pathway, and becomes N-glycosylated before entering the chloroplast.     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.     

Structural characterization of a serendipitously discovered bioactive macromolecule, lignin sulfate

The herpes simplex virus-1 (HSV-1) utilizes cell-surface glycosaminoglycan, heparan sulfate, to gain entry into cells and cause infection. In a search for synthetic mimics of heparan sulfate to prevent HSV infection, we discovered potent inhibitory activity arising from sulfation of a monomeric flavonoid. Yet, detailed screening indicated that the sulfated flavonoid was completely inactive and the potent inhibitory activity arose from a macromolecular substance present in the parent flavonoid. The active principle was identified through a battery of biophysical and chemical analyses as a sulfated form of lignin, a three-dimensional network polymer composed of substituted phenylpropanoid monomers. Mass spectral analysis of the parent lignin and its sulfated derivative indicates the presence of p-coumaryl monomers interconnected through uncondensed beta-O-4-linkages. Elemental analysis of lignin sulfate correlates primarily with a polymer of p-coumaryl alcohol containing one sulfate group. High-performance size exclusion chromatography shows a wide molecular weight distribution from 1.5 to 40 kDa suggesting significant polydispersity. Polyacrylamide gel electrophoresis (PAGE) analysis indicates a highly networked polymer that differs significantly from linear charged polymers with respect to its electrophoretic mobility. Overall, macromolecular lignin sulfate presents a multitude of substructures that can interact with biomolecules, including viral glycoproteins, using hydrophobic, hydrogen-bonding, and ionic forces. Thus, lignin sulfate represents a large number of interesting structures with potential medicinal benefits.     

Purified Wnt-5a increases differentiation of midbrain dopaminergic cells and dishevelled phosphorylation

The Wnt family of lipoproteins regulates several aspects of the development of the nervous system. Recently, we reported that Wnt-3a enhances the proliferation of midbrain dopaminergic precursors and that Wnt-5a promotes their differentiation into dopaminergic neurones. Here we report the purification of hemagglutinin-tagged Wnt-5a using a three-step purification method similar to that previously described for Wnt-3a. Haemagglutinin-tagged Wnt-5a was biologically active and induced the differentiation of immature primary midbrain precursors into tyrosine hydroxylase-positive dopaminergic neurones. Using a substantia nigra-derived dopaminergic cell line (SN4741), we found that Wnt-5a, unlike Wnt-3a, did not promote beta-catenin phosphorylation or stabilization. However, both Wnt-5a and Wnt-3a activated dishevelled, as assessed by a phosphorylation-dependent mobility shift. Moreover, the activity of Wnt-5a on dishevelled was blocked by pre-treatment with acyl protein thioesterase-1, indicating that palmitoylation of Wnt-5a is necessary for its function. Thus, our results suggest that Wnt-3a and Wnt-5a, respectively, activate canonical and non-canonical Wnt signalling pathways in ventral midbrain dopaminergic cells. Furthermore, we identify dishevelled as a key player in transducing both Wnt canonical and non-canonical signals in dopaminergic cells.     

Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum. acid-base transporters; cystic fibrosis transmembrane conductance regulator channel; surface pH gradient; mucus gels; trefoil peptides     

Pre-diabetes and diabetes are independently associated with adverse cognitive test results: a cross-sectional,population-based study

Background

Diabetes is a risk factor for cognitive impairment, but whether there is also a link between pre-diabetes and cognitive dysfunction is not yet fully established. The aim of this observational study was to investigate associations between pre-diabetes/diabetes and cognitive test results, and also between glucose levels measured during the Oral Glucose Tolerance Test (OGTT) and cognitive outcomes.

Methods

During 2007–2012, in all 2994 people (mean age 72?years), residing in Malmö, Sweden, underwent a clinical examination including the OGTT, cardiovascular measurements including carotid-femoral pulse wave velocity (c-f PWV) and two cognitive tests, the Mini Mental State Examination (MMSE), measuring global cognitive function, and A Quick Test of Cognitive Speed (AQT), measuring processing speed and executive functioning. Regression analyses were performed to investigate associations between: (a) categories of normal or impaired glucose metabolism, and (b) OGTT measurements, respectively, as exposure variables and cognitive test results as outcomes. Adjustments were made for demographics, lifestyle factors and cardiovascular risk factors.

Results

Participants with pre-diabetes and diabetes scored slightly worse cognitive test results compared to the control group. Results of participants with a long disease duration of diabetes since the baseline examination 13?years earlier were poorer (mean AQT test time 17.8?s slower than controls, p?<?0.001). Linear associations were found between fasting and 2-h glucose and cognitive outcomes in the whole population, but also in a sub-analysis including only individuals without diabetes (for 2-h glucose and MMSE results: B?=???2.961, p?=?0.005). Associations were stronger for older or less physically active individuals. When adjusting for cardiovascular risk factors, most correlations were non-significant.

Conclusions

Pre-diabetes and diabetes are associated with minor deficits in global cognitive function, processing speed and executive functioning. Long-standing diabetes is associated with bigger deficits. There appears to be a continuous inverse correlation between glucose levels and cognitive test results, also for people without diabetes. Associations are stronger in older and less physically active individuals. Cardiovascular factors are important mediating factors in the pathway between diabetes and cognitive dysfunction.

     

The effect of polyvinyl alcohol and polyvinyl pyrrolidone on diffusion artifacts in lactate dehydrogenase histochemistry

Summary The effect of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), alone and in combination, on diffusion artifacts in histochemical incubations has been investigated using LDH as model enzyme. By measuring the amount of formazan in the medium at the end of the incubation it has been shown that both substances, but especially PVA, are effective in limiting diffusion. The significance of this is discussed in general as well as in relation to other procedures used to reduce diffusion artifacts.The Following Abbreviations are used in the Article NAD -Nicotinamide Adenine Dinucleotide - NADH -Nicotinamide Adenine Dinucleotide, Reduced form - PVA Polyvinyl alcohol - PVP Polyvinyl pyrrolidone - PMS Phenazine methosulfate - tris tris (hydroxymethyl)-aminomethan - Nitro-BT Nitro Blue Tetrazolium - LDH Lactic dehydrogenase     

Autofluorescent granules in cells of human dermis

Summary The results of a continued investigation on dermal autofluorescent pigment cells are presented. Previously obtained histochemical data were suggestive of a lipo-pigment component of the fluorescent cytoplasmic granules. Spectrophotofluorometric results obtained in the present study seem to confirm this supposition. Electron microscopic observations on the cells and especially their granules show certain similarites between the fluorescent dermal pigment and lipofuscin and/or lysosomal structures. It cannot be excluded that some type of melanin is present in the granules. The findings are discussed in the light of modern views on melanins and lipo-pigments. Possible functions of the cells as well as their relation to epidermal and dermal elements are considered.This investigation has been supported by grants from the Swedish Government (Reser-vationsanslaget), Stiftelsen Therese och Johan Anderssons Minne, Svenska Sällskapet for medicinsk forskning and the Wallenberg Foundation.     

Autofluorescent granules in cells of human dermis

Summary Cells with autofluorescent granules are common in the dermal connective tissue of human skin. The cytoplasmic granules appear to be of lipo-pigment nature. The cells show phagocytic properties and it can therefore not be excluded that the cytoplasmic granular structures are ingested material. There are certain similarities between the observed dermal autofluorescent cells (DAF-cells) and chromatophores (melanophages) of the dermis. Convincing histochemical evidence has not been obtained for the presence of catecholamines in these fluorescent cells, which has been suggested.This investigation has been supported by grants from the Swedish Government (Reservationsanslaget), Stiftelsen Therese och Johan Anderssons Minne, the U.S. Public Health Service (Grant No. C4716) and the School of Aerospace Medicine A.F.S.C. through the European Office, Aerospace Research U.S.A.F.