ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons

Induced pluripotent stem cells (iPSCs) hold promise to revolutionize studies of intracellular transport in live human neurons and to shed new light on the role of dysfunctional transport in neurodegenerative disorders. Here, we describe an approach for live imaging of axonal and dendritic transport in iPSC‐derived cortical neurons. We use transfection and transient expression of genetically‐encoded fluorescent markers to characterize the motility of Rab‐positive vesicles, including early, late and recycling endosomes, as well as autophagosomes and mitochondria in iPSC‐derived neurons. Comparing transport parameters of these organelles with data from primary rat hippocampal neurons, we uncover remarkable similarities. In addition, we generated lysosomal‐associated membrane protein 1 (LAMP1)‐enhanced green fluorescent protein (EGFP) knock‐in iPSCs and show that knock‐in neurons can be used to study the transport of endogenously labeled vesicles, as a parallel approach to the transient overexpression of fluorescently labeled organelle markers.     

Variations in enzyme activity in stomach and pancreatic tissue and digesta in piglets around weaning

A study was performed to investigate the effect of weaning at 4 weeks of age on the activity of digestive enzymes in the stomach and pancreatic tissue and in digesta from 3 days prior to weaning to 9 days postweaning in 64 piglets. In stomach tissue the activity of pepsin and gastric lipase was determined. Pepsin activity declined abruptly after weaning but 5 days postweaning the weaning level was regained and in the gastric contents no change in pepsin activity was observed. Weaning did not influence the activity of gastric lipase. The activity of eight enzymes and a cofactor was measured in pancreatic tissue. The effect of weaning on the enzyme activity was highly significant for all enzymes except elastase. The activity of all enzymes remained at the weaning level during day 1–2 postweaning followed by a reduction of the activity. The activity of trypsin, carboxypeptidase A, amylase and lipase exhibited minimum activity 5 days postweaning. Trypsin activity increased to the preweaning level on day 7–9 whereas the activity of the others increased but did not reach the preweaning level. The activity of chymotrypsin, carboxypeptidase B and carboxyl ester hydrolase decreased during the entire experimental period. In digesta no effect of weaning was observed on the activity of amylase and trypsin. The activity of chymotrypsin was reduced after weaning in the proximal third of the small intestine and lipase and carboxyl ester hydrolase activity was reduced in the middle and distal parts of the small intestine after weaning. The present study shows that the activities of the digestive enzymes in the pancreatic tissue are affected by weaning. Even though the pancreatic secretion cannot be judged from these results they show that the enzymes respond differently to weaning. In general the activity of the digestive enzymes in pancreatic tissue is low on day 5 postweaning which in interaction with other factors may increase the risk of developing postweaning diarrhoea.     

Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.     

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.     

An in situ evidence for autocrine function of NO in the vasculature

The concept of endothelium derived relaxing factor (EDRF) implies that nitric oxide (NO) generated by NO synthase in the endothelium diffuses to the underlying vascular smooth muscle cells (VSMC) modulating thereby vascular tone. VSMC were regarded as passive recipients of NO from endothelial cells. However, this paradigm of a paracrine function of NO became currently subject to considerable debate. To address this issue, we examined the localization of enzymes engaged in l-arginine-NO-cGMP signaling in the rat blood vessels. Employing multiple immunocytochemical labeling complemented with signal amplification, electron microscopy, Western blotting, and RT-PCR, we found that NO synthase was differentially expressed in blood vessels depending on the blood vessel type. Moreover, the expression pattern of NO synthase in VSMC showed striking parallels with arginase and soluble guanylyl cyclase. Our findings challenge the commonly accepted view that the expression of NO synthase is restricted to vascular endothelial cells and lends further support to an alternative mechanism, by which constitutive local NOS expression in VSMC may modulate vascular functions in an endothelium-independent manner. Moreover, the co-expression of enzymes engaged in l-arginine-NO-cGMP signaling (NO synthase, arginase, and soluble guanylyl cyclase) in VSMC is indicative of an autocrine fashion of NO signaling in the vasculature in addition to the paracrine role of NO generated in the endothelium.     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Zur Tagesaktivit?t der Seeschwalben

Zusammenfassung Zählungen überhinfliegender und an- und abfliegender Fluß- und Küstenseeschwalben auf der Insel Wangerooge ergaben eine deutliche Tageszeitbeziehung, jedoch keine Gezeitenabhängigkeit, ganz im Gegensatz zu den Beobachtungen an fischenden, fütternden und rastenden Seeschwalben. Die Ursache wird darin gesehen, daß bei den Zählungen fliegender Vögel vornehmlich solche Tiere erfaßt werden, die sich an sexuellen oder sozialen Flügen beteiligen. Diese werden offenbar in ihrer Häufigkeit und Intensität stark von der Tageszeit beeinflußt. Die bei diesen Zählungen miterfaßten Nahrungsflüge treten demgegenüber zahlenmäßig so stark zurück, daß ihre Gezeitenabhängigkeit im Zählergebnis nicht zum Ausdruck kommt. Damit wurde gezeigt, wie innerhalb einer Seeschwalbenkolonie verschiedene Verhaltensweisen der Vögel vorwiegend entweder von der Tageszeit oder vom Gezeitenrhythmus beeinflußt werden können.

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Summary On the island of Wangerooge counting of Common and Arctic Terns flying over the colony and starting and landing at their nests, showed a remarkable relation to daytime but not to tidal phenomena, in contrast to observations on terns that were fishing, feeding or resting on the ground. The author could show that especially such birds are included in the counting of flying terns, which take part in sexual or social flights. Frequency and intensity of these flights become affected by daytime. On the other hand the number of flights performed by birds flying for food or returning from their fishinggrounds is so unimportant, that the results of the counting cannot show their relation to tidal phenomena. By his observations the author could prove that different elements of the behaviour of the terns observed within one colony could become affected either by daytime or by the tides.