Elevated Golgi pH impairs terminal N‐glycosylation by inducing mislocalization of Golgi glycosyltransferases

Acidic pH of the Golgi lumen is known to be crucial for correct glycosylation, transport and sorting of proteins and lipids during their transit through the organelle. To better understand why Golgi acidity is important for these processes, we have examined here the most pH sensitive events in N‐glycosylation by sequentially raising Golgi luminal pH with chloroquine (CQ), a weak base. We show that only a 0.2 pH unit increase (20 µM CQ) is sufficient to markedly impair terminal α(2,3)‐sialylation of an N‐glycosylated reporter protein (CEA), and to induce selective mislocalization of the corresponding α(2,3)‐sialyltransferase (ST3) into the endosomal compartments. Much higher pH increase was required to impair α(2,6)‐sialylation, or the proximal glycosylation steps such as β(1,4)‐galactosylation or acquisition of Endo H resistance, and the steady‐state localization of the key enzymes responsible for these modifications (ST6, GalT I, MANII). The overall Golgi morphology also remained unaltered, except when Golgi pH was raised close to neutral. By using transmembrane domain chimeras between the ST6 and ST3, we also show that the luminal domain of the ST6 is mainly responsible for its less pH sensitive localization in the Golgi. Collectively, these results emphasize that moderate Golgi pH alterations such as those detected in cancer cells can impair N‐glycosylation by inducing selective mislocalization of only certain Golgi glycosyltransferases. J. Cell. Physiol. 220: 144–154, 2009. © 2009 Wiley‐Liss, Inc.     

CGHpower: exploring sample size calculations for chromosomal copy number experiments

Background  

Determining a suitable sample size is an important step in the planning of microarray experiments. Increasing the number of arrays gives more statistical power, but adds to the total cost of the experiment. Several approaches for sample size determination have been developed for expression array studies, but so far none has been proposed for array comparative genomic hybridization (aCGH).     

Nitrogen retention by peatland buffer areas at six forested catchments in southern and central Finland

We studied the nitrogen retention capacity of six peatland buffer areas constructed in forested catchments in southern and central Finland. The buffers (0.1–4.9% of the total catchment area) were either undrained mires or drained peatlands rewetted 4–7 years before the present study. The N retention capacity was studied by adding ammonium nitrate (NH₄NO₃–N) solution into the inflow waters of the buffers once (one area) or twice (five areas) during a period of 4–6 years. Except for the first N addition in one area, the three largest buffer areas (relative size > 1%) retained the added inorganic N almost completely; their retention efficiencies during the year of addition were >93% for both NO₃–N and NH₄–N. Two of the three small buffers (relative size < 0.25%) were also able to reduce inorganic N from the through-flow waters effectively; their retention capacities for inorganic nitrogen varied between 58 and 89%. However, one small buffer area had a retention capacity of only <20%. The factors contributing to efficient N retention were hydrological load during N addition, relative size of the buffer area, and its length, i.e. the distance between the inflow and outflow points. If there was any release of the added N, it mostly occurred within a relatively short-time period (<100 days) after the treatment. The buffer areas appeared to be efficient and long-term sinks for inorganic nitrogen because the release of N during the 2–4 years after N addition was minor.     

Distinct differentiation characteristics of individual human embryonic stem cell lines

Background  

Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic.     

Early-onset renal cell carcinoma as a novel extraparaganglial component of SDHB-associated heritable paraganglioma

Hereditary paraganglioma syndrome has recently been shown to be caused by germline heterozygous mutations in three (SDHB, SDHC, and SDHD) of the four genes that encode mitochondrial succinate dehydrogenase. Extraparaganglial component neoplasias have never been previously documented. In a population-based registry of symptomatic presentations of phaeochromocytoma/paraganglioma comprising 352 registrants, among whom 16 unrelated registrants were SDHB mutation positive, one family with germline SDHB mutation c.847-50delTCTC had two members with renal cell carcinoma (RCC), of solid histology, at ages 24 and 26 years. Both also had paraganglioma. A registry of early-onset RCCs revealed a family comprising a son with clear-cell RCC and his mother with a cardiac tumor, both with the germline SDHB R27X mutation. The cardiac tumor proved to be a paraganglioma. All RCCs showed loss of the remaining wild-type allele. Our observations suggest that germline SDHB mutations can predispose to early-onset kidney cancers in addition to paragangliomas and carry implications for medical surveillance.     

Defective acidification of intracellular organelles results in aberrant secretion of cathepsin D in cancer cells

Aberrant secretion of lysosomal hydrolases such as (pro)cathepsin D (proCD) is a common phenotypic change in many human cancers. Here we explore the underlying molecular defect(s) and find that MCF-7 breast and CaCo-2 colorectal cancer cells that are unable to acidify their endosomal compartments secreted higher amounts of proCD than did acidification-competent cancer cell types. The latter secreted equivalent amounts of proCD only after dissipation of their organellar pH gradients with NH(4)Cl. Assessing the critical steps that resulted in proCD secretion revealed that the Golgi-associated sorting receptor for CD, i.e. the cation-independent mannose-6-phosphate receptor (MPR300), was aberrantly distributed in acidification-defective MCF-7 cells. It accumulated mainly in late endosomes and/or lysosomes as a complex with its ligand (proCD or intermediate CD), as evidenced by its co-localization with both CD and LAMP-2, a late endosome/lysosome marker. Our immunoprecipitation analyses also showed that MCF-7 cells possessed 7-fold higher levels of receptor-enzyme complexes than did acidification-competent cells. NH(4)Cl induced similar receptor redistribution into LAMP-2-positive structures in acidification-competent cells but not in MCF-7 cells. The receptor also recovered its normal Golgi localization upon drug removal. Based on these observations, we conclude that defective acidification results in the aberrant secretion of proCD in certain cancer cells and interferes mainly with the normal disassembly of the receptor-enzyme complexes and efficient receptor reutilization in the Golgi.     

Identification of the streptococcal M protein binding site on membrane cofactor protein (CD46)

Adherence of group A streptococcus (GAS) to keratinocytes is mediated by an interaction between human CD46 (membrane cofactor protein) with streptococcal cell surface M protein. CD46 belongs to a family of proteins that contain structurally related short consensus repeat (SCR) domains and regulate the activation of the complement components C3b and/or C4b. CD46 possesses four SCR domains and the aim of this study was to characterize their interaction with M protein. Following confirmation of the M6 protein-dependent interaction between GAS and human keratinocytes, we demonstrated that M6 protein binds soluble recombinant CD46 protein and to a CD46 construct containing only SCRs 3 and 4. M6 protein did not bind to soluble recombinant CD46 chimeric proteins that had the third and/or fourth SCR domains replaced with the corresponding domains from another complement regulator, CD55 (decay-accelerating factor). Homology-based molecular modeling of CD46 SCRs 3 and 4 revealed a cluster of positively charged residues between the interface of these SCR domains similar to the verified M protein binding sites on the plasma complement regulators factor H and C4b-binding protein. The presence of excess M6 protein did not inhibit the cofactor activity of CD46 and the presence of excess C3b did not inhibit the ability of CD46 to bind M6 protein by ELISA. In conclusion, 1) adherence of M6 GAS to keratinocytes is M protein dependent and 2) a major M protein binding site is located within SCRs 3 and 4, probably at the interface of these two domains, at a site distinct from the C3b-binding and cofactor site of CD46.     

Cellular phone use does not acutely affect blood pressure or heart rate of humans

A recent study raised concern about increase of resting blood pressure after a 35 min exposure to the radiofrequency (RF) field emitted by a 900 MHz cellular phone. In this randomized, double blind, placebo controlled crossover trial, 32 healthy subjects were submitted to 900 MHz (2 W), 1800 MHz (1 W) cellular phone exposure, and to sham exposure in separate sessions. Arterial blood pressure (arm cuff method) and heart rate were measured during and after the 35 min RF and sham exposure sessions. We evaluated cardiovascular responses in terms of blood pressure and heart rate during controlled breathing, spontaneous breathing, head-up tilt table test, Valsalva manoeuvre and deep breathing test. Arterial blood pressure and heart rate did not change significantly during or after the 35 min RF exposures at 900 MHz or 1800 MHz, compared to sham exposure. The results of this study indicate that exposure to a cellular phone, using 900 MHz or 1800 MHz with maximal allowed antenna powers, does not acutely change arterial blood pressure and heart rate.     

The existence of a hypnotic state revealed by eye movements

Hypnosis has had a long and controversial history in psychology, psychiatry and neurology, but the basic nature of hypnotic phenomena still remains unclear. Different theoretical approaches disagree as to whether or not hypnosis may involve an altered mental state. So far, a hypnotic state has never been convincingly demonstrated, if the criteria for the state are that it involves some objectively measurable and replicable behavioural or physiological phenomena that cannot be faked or simulated by non-hypnotized control subjects. We present a detailed case study of a highly hypnotizable subject who reliably shows a range of changes in both automatic and volitional eye movements when given a hypnotic induction. These changes correspond well with the phenomenon referred to as the "trance stare" in the hypnosis literature. Our results show that this 'trance stare' is associated with large and objective changes in the optokinetic reflex, the pupillary reflex and programming a saccade to a single target. Control subjects could not imitate these changes voluntarily. For the majority of people, hypnotic induction brings about states resembling normal focused attention or mental imagery. Our data nevertheless highlight that in some cases hypnosis may involve a special state, which qualitatively differs from the normal state of consciousness.