Short-term parenteral application of α-tocopherol leads to increased concentration in plasma and tissues of the rat

Numerous studies suggest that supplemental vitamin E prior to or during vast surgeries might diminish or even prevent ischemia/reperfusion-induced injuries. In the present placebo-controlled study male Sprague-Dawley rats were supplemented parenterally or orally with α-tocopherol for three consecutive days. The applied amount of α-tocopherol was 2.3 μmol per day for oral and 1.2 μmol per day for parenteral supplementation. The enrichment of vitamin E concentrations in plasma and tissue samples (aortic endothelium, liver, and lung) was determined by HPLC. The vitamin E level was elevated following intravenous supplementation in plasma (21.4±1.9 μmol/L vs. 10.2±1.7 μmol/L in parenteral control group), in aortic endothelium (1.1±0.2 pmol/mm² vs. 0.5±0.1 pmol/mm²) and in liver and lung (41.3±7.5 pmol/mg vs. 22.9±6.5 pmol/mg and 75.6±13.6 pmol/mg vs. 51.7±5.9 pmol/mg, respectively). Oral supplementation for three days also led to an increased level in liver (38.2±7.7 pmol/mg vs. 22.9±6.6 pmol/mg in oral control group) and in lung (67.8±5.7 pmol/mg vs. 51.7±9.3 pmol/mg) but not in aortic endothelium or plasma (0.8±0.3 pmol/mm² vs. 0.6±0.3 pmol/mm² and 12.0±2.2 μmol/L vs. 10.7±2.6 μol/L.)     

What keeps cells in tissues behaving normally in the face of myriad mutations?

The use of a reporter gene in transgenic mice indicates that there are many local mutations and large genomic rearrangements per somatic cell that accumulate with age at different rates per organ and without visible effects. Dissociation of the cells for monolayer culture brings out great heterogeneity of size and loss of function among cells that presumably reflect genetic and epigenetic differences among the cells, but are masked in organized tissue. The regulatory power of a mass of contiguous normal cells is expressed in its capacity to normalize the appearance and growth behavior of solitary homophilic neoplastic cells, and to redirect differentiation of solitary heterophilic stem-like cells. Intimate contact between the interacting cells is required to induce these changes. The normalization of the neoplastic phenotype does not require gap junctional communication between cells, though transdifferentiation might. These varied relationships are manifestations of the unifying biological principle of "order in the large over heterogeneity in the small".     

Does heparin occur in mucosal mast cells of the rat small intestine?

Microspectrophotometric analyses combined with model experiments using polyacrylamide films containing different types of purified glycosaminoglycan or glycosaminoglycan and basic protein, have been applied to investigate the chemical characteristics of the glycosaminoglycan moiety present in rat mucosal mast cell granules. The results obtained with these histochemical techniques present evidence of the absence of significant amounts of heparin and the presence of lower sulfated glycosaminoglycan(s) in the granules of these cells.     

Acute cholesterol depletion leads to net loss of the organic osmolyte taurine in Ehrlich Lettré tumor cells

In mammalian cells, the organic osmolyte taurine is accumulated by the Na-dependent taurine transporter TauT and released though the volume- and DIDS-sensitive organic anion channel. Incubating Ehrlich Lettré tumor cells with methyl-β-cyclodextrin (5 mM, 1 h) reduces the total cholesterol pool to 60 ± 5% of the control value. Electron spin resonance data indicate a concomitant disruption of cholesterol-rich micro-domains. Active taurine uptake, cellular taurine content, and cell volume are reduced by 50, 20 and 20% compared to control values, respectively, whereas the passive taurine release is increased 4.5-fold under isotonic conditions following cholesterol depletion. However, taurine release under isotonic conditions is insensitive to DIDS and inhibitors of the volume-regulated anion channel. Uptake and release of meAIB are similarly affected following cholesterol depletion. Kinetic analysis reveals that cholesterol depletion increases TauT’s affinity toward taurine but reduces its maximal transport capacity. Cholesterol depletion has no impact on TauT regulation by protein kinases A and C. Phospholipase A₂ activity, which is required for the activation of volume-sensitive organic anion channel (VSOAC), is increased under isotonic and hypotonic conditions following cholesterol depletion, whereas taurine release under hypotonic conditions is reduced following cholesterol depletion. Hence, acute cholesterol depletion of Ehrlich Lettré cells leads to reduced TauT and VSOAC activities and at the same time increases the release of organic osmolytes via a leak pathway different from the volume-sensitive pathways for amino acids and anions.     

Variation in the methods leads to variation in the interpretation of biodiversity–ecosystem multifunctionality relationships

生物多样性常常和生态系统多功能性(生态系统同时提供多个生态系统功能的能力)正相关。然而,生物多样性与生态系统多功能性的关系是否依赖于生态系统功能的数目有诸多争议。其中,生物多样性对生态系统多功能性的影响或许不随生态系统功能数目的变化而变化,或者随生态系统功能数目的增多而增强。我们期望通过研究不同生态系统多功能性指数的统计原理来解决这些争议。 我们使用了模型模拟和一系列来自不同空间尺度(从局域到全球)和不同生物群系(温带和高寒草地、森林和干旱地)的经验数据。我们回顾了量化生态系统多功能性的三种方法,包括平均值法、加和法和阈值法。我们发现随着生态系统功能数目的增加,生物多样性与生态系统多功能性的关系要么不变,要么增强。这些结果可由平均和加和的多功能性指数的统计原理来解释。具体来讲,当利用生态系统功能的平均值计算多功能性指数时,由于多样性对多功能性的效应等于多样性对单个生态系统功能效应的平均值,所以不会随生态系统功能数目的变化而变化。同样的道理,当利用单个生态系统的加和值计算多功能性指数时,多样性的效应会随着生态系统功能数目的增加而增强。我们提出了一个改进的多功能性指数,将平均或加和多功能性指数转化为标准化的多功能性指数, 以便于对不同研究的结果进行比较。此外,我们提出了基于变量数值范围的标准化方法来解决阈值法的数学假象问题(多样性效应随生态系统功能数目的增加而增强)。我们的研究结果表明,量化多功能性指数的方法不同,结果也不同。因此,有必要加深对不同方法数理基础的理解。而标准化的多功能性指数为比较不同研究中的生物多样性与生态系统多功能性的关系提供了有效的方法。     

Functional morphology of mucosal goblet cells based on spatial separation of orifice openings to the surface – Application to the rabbit bulbar conjunctiva

The purpose of the study was to assess spatial separation of goblet cell orifices observed at the surface of the rabbit bulbar conjunctiva by scanning electron microscopy (SEM) specimens of the bulbar conjunctiva from 8 healthy pigmented rabbits were obtained using a special preparation technique by which the tissue was carefully stretched out during glutaraldehyde fixation. On high magnification prints of SEM images of the conjunctival surface, the locations of goblet cell openings (orifices) to the apical surface were marked and the centre-to-centre spacing between all such orifices measured. Across the regions of interest (ROI), with averaged dimensions of 322 μm × 230 μm (adjusted for tissue shrinkage), the averaged value for the distances between all orifices was 196 μm (range 141–241 μm), with the calculated density of orifices being 412 mm⁻². A sequential order-based analysis of the spatial separation between orifices indicated a predictable value of 6 μm, a separation that showed a nearly linear inter-dependence over distances of at least 200 μm. The openings of goblet cells to the surface of unstimulated bulbar conjunctiva have a organized spatial distribution that is consistent with there being an organized control of goblet cell secretion.     

Metabolism of the plant sulfolipid—Sulfoquinovosyldiacylglycerol: Degradation in animal tissues

Metabolism of the plant sulfolipid—sulfoquinovosyldiacylglycerol (SQDG)—was studied in animal tissues. In vivo experiments with [³⁵S]SQDG in guinea pigs showed that this lipid is not absorbed intact in the gastrointestinal tract. In these experiments, 3 h after administration of [³⁵S]SQDG, the intestinal mucosa contained 1 to 5% of the radioactivity as SQDG, while the remainder was in a water-soluble form. Analysis of the water-soluble components showed that about 60% of the radioactivity was present as sulfoquinovosylglycerol (SQG) and the remainder was present as free SO²⁻₄. In the blood, 99% of the radioactivity was present as SO²⁻₄, SQG was not observed. In liver, only very little radioactivity was observed and appeared to be mainly in the form of SO²⁻₄. Experiments with everted intestinal sacs of guinea pigs confirmed the formation of SQG, SO²⁻₄, and, in addition, sulfoquinovosylmonoacylglycerol (SQMG) in this tissue. In vitro experiments with saline extracts of acetone powders of pancreas and intestinal mucosa of guinea pig, sheep, and rat showed that [³⁵S]SQDG was deacylated to SQMG (sulfolipase A activity) and SQG (sulfolipase B activity). It is concluded that animal tissues deacylate SQDG in a stepwise manner to SQG. It is further metabolized to yield free SO²⁻₄ by cleavage of the C-S bond which appears to be brought about by the intestinal microflora. Sheep pancreatic sulfolipases were characterized. Bile salts, sodium dodecyl sulfate, and Triton X-100 inhibited the pancreatic sulfolipases, while CaCl₂ activated them. Substrate competition experiments and investigations on substrate specificity with a partially purified preparation indicated that relatively specific sulfolipase(s) may exist in pancreas. Among the species tested, guinea pig tissues showed the highest sulfolipase A and B activities followed sheep and rat tissues. Pancreatic enzymes were 18 to 60 times more active than intestinal enzymes.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

On the role of peroxisomes in the metabolism of lipids — evidence from studies on mammalian tissues in vivo

Summary Recent investigations into the role of peroxisomes in mammalian lipid metabolism have employed double isotope methodologies to examine the influence of peroxisomal agents on lipid turnover in the liver and extra hepatic tissues of the living animal.The action of these agents, all of which caused extensive changes in the flux of lipid metabolism in the treated animals, may best be viewed in relation to their effects on the common pathway of fatty acid oxidation in peroxisomes.Clofibrate, for example, acts through induction of peroxisomal oxidases and catalase; glycolate and ethanol through activation of this pathway; and aminotriazole and allylisopropylacetamide through inhibition of the catalase step in the sequence.The data from these studies provide support for the concept of an important contributory and regulatory role of peroxisomes in relation to the overall balance of lipid metabolism, and emphasize that these organelles play a significant role in the oxidation of common fatty acids, as well as a potential for the elimination of fatty acids that are poorly oxidized by mitochondria.Additionally, the data raise intriguing questions on the extension of peroxisomal influence to include phospholipid metabolism and the substantial degree of inter-tissue communication which is involved in the balance of lipid metabolism in the whole animal.     

Activation of activin type IB receptor signals in pancreatic β cells leads to defective insulin secretion through the attenuation of ATP-sensitive K channel activity

In studies of gene-ablated mice, activin signaling through activin type IIB receptors (ActRIIB) and Smad2 has been shown to regulate not only pancreatic β cell mass but also insulin secretion. However, it still remains unclear whether gain of function of activin signaling is involved in the modulation of pancreatic β cell mass and insulin secretion. To identify distinct roles of activin signaling in pancreatic β cells, the Cre-loxP system was used to activate signaling through activin type IB receptor (ActRIB) in pancreatic β cells. The resultant mice (pancreatic β cell-specific ActRIB transgenic (Tg) mice; ActRIBCAβTg) exhibited a defect in glucose-stimulated insulin secretion (GSIS) and a progressive impairment of glucose tolerance. Patch-clamp techniques revealed that the activity of ATP-sensitive K⁺ channels (K_ATP channels) was decreased in mutant β cells. These results indicate that an appropriate level of activin signaling may be required for GSIS in pancreatic β cells, and that activin signaling involves modulation of K_ATP channel activity.     

Production of α2,6-sialylated IgG1 in CHO cells

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG''s effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc''s amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.     

Production of α2,6-sialylated IgG1 in CHO cells

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.     

Immediate stoichiometric appearance of β-galactosidase products in the medium of Escherichia coli cells incubated with lactose

Products of β-galactosidase action on lactose by intact E. coli cells appeared in the medium as soon as lactose was added and the amount of product was equal to the lactose used. No detectable levels of β-galactosidase were found in the medium and lactose was not significantly broken down unless lac permease was present. The appearance did not depend upon the presence of any of the commonly known galactose or glucose permease systems. The Km of product appearance from whole cells was equal to the K_t for lactose transport by lac permease. When the cells were broken the Km became the normal β-galactosidase Km.