Development and advantages of serum-free, chemically defined nutrient media for culturing of nerve tissue

This paper describes in a historical perspective the development of serum-free nutrient media suitable for long-term culturing of nerve tissue. Several disadvantages of the use of serum are discussed, coupled with an acknowledgement that it is not always advisable to replace a routinely used serum-supplemented medium by a chemically defined medium with the expectation of immediate success. Therefore a strategy is given on how to develop a chemically defined medium that is thoroughly tuned to the specific needs of the cell type to be cultured. It is argued that such a medium has several substantial advantages over the use of serum.     

The influence of some sympatholytic,parasympatholytic and serotonin-synthesis-inhibiting agents on the ultrastructure of the rabbit pineal organ

The influence of certain drugs on the ultrastructure of rabbit pinealocytes was studied. The results obtained after administration of p-chlorophenylalanine and p-chloroamphetamine support the hypothesis proposed earlier that the smooth endoplasmic reticulum in the light pinealocytes is involved in indoleamine synthesis. The administration of either one of the sympatholytic agents, 6-hydroxydopamine or alpha-methyl-p-tyrosine, induced typical fine structural changes corresponding to those observed after surgical sympathectomy.     

Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix

Timely sensing of lipopolysaccharide (LPS) is critical for the host to fight invading Gram-negative bacteria. We recently showed that apolipoprotein CI (apoCI) (apoCI_1–57) avidly binds to LPS, involving an LPS-binding motif (apoCI_48–54), and thereby enhances the LPS-induced inflammatory response. Our current aim was to further elucidate the structure and function relationship of apoCI with respect to its LPS-modulating characteristics and to unravel the mechanism by which apoCI enhances the biological activity of LPS. We designed and generated N- and C-terminal apoCI-derived peptides containing varying numbers of alternating cationic/hydrophobic motifs. ApoCI_1–38, apoCI_1–30, and apoCI_35–57 were able to bind LPS, whereas apoCI_1–23 and apoCI_46–57 did not bind LPS. In line with their LPS-binding characteristics, apoCI_1–38, apoCI_1–30, and apoCI_35–57 prolonged the serum residence of ¹²⁵I-LPS by reducing its association with the liver. Accordingly, both apoCI_1–30 and apoCI_35–57 enhanced the LPS-induced TNFα response in vitro (RAW 264.7 macrophages) and in vivo (C57Bl/6 mice). Additional in vitro studies showed that the stimulating effect of apoCI on the LPS response resembles that of LPS-binding protein (LBP) and depends on CD14/ Toll-like receptor 4 signaling. We conclude that apoCI contains structural elements in both its N-terminal and C-terminal helix to bind LPS and to enhance the proinflammatory response toward LPS via a mechanism similar to LBP.     

Dynamics of insulin signalling in liver during hyperinsulinemic euglycaemic clamp conditions in vivo and the effects of high-fat feeding in male mice

Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.     

Molecular Architecture of the Centriole Proteome: The Conserved WD40 Domain Protein POC1 Is Required for Centriole Duplication and Length Control

Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data suggest that a large number of proteins besides tubulin are necessary for the formation and maintenance of a centriole''s complex structure. Expansion of the preexisting centriole proteome from the green alga Chlamydomonas reinhardtii revealed additional human disease genes, emphasizing the significance of centrioles in normal human tissue homeostasis. We found that two classes of ciliary disease genes were highly represented among the basal body proteome: cystic kidney disease (especially nephronophthisis) syndromes, including Meckel/Joubert-like and oral-facial-digital syndrome, caused by mutations in CEP290, MKS1, OFD1, and AHI1/Jouberin proteins and cone-rod dystrophy syndrome genes, including UNC-119/HRG4, NPHP4, and RPGR1. We further characterized proteome of the centriole (POC) 1, a highly abundant WD40 domain-containing centriole protein. We found that POC1 is recruited to nascent procentrioles and localizes in a highly asymmetrical pattern in mature centrioles corresponding to sites of basal-body fiber attachment. Knockdown of POC1 in human cells caused a reduction in centriole duplication, whereas overexpression caused the appearance of elongated centriole-like structures. Together, these data suggest that POC1 is involved in early steps of centriole duplication as well as in the later steps of centriole length control.     

Interferon-gamma has immunomodulatory effects with minor endocrine and metabolic effects in humans

To evaluatewhether interferon- (IFN-) is involved in the interaction betweenthe immune and endocrine systems in vivo, we studied six healthysubjects twice in a placebo-controlled trial: once after administrationof recombinant human IFN- and, on another occasion, afteradministration of saline. The rate of appearance of glucose wasdetermined by infusion of[6,6-²H₂]glucoseand resting energy expenditure by indirect calorimetry. Human leukocyteantigen-DR gene expression on monocytes and serum neopterin increased after administration of IFN-(P < 0.05 vs. control). IFN-increased serum interleukin-6 levels significantly. Levels of tumornecrosis factor- remained below detection limits. IFN- increasedplasma concentrations of ACTH and cortisol(P < 0.05 vs. control), IFN- didnot alter concentrations of growth hormone,(nor)epinephrine, insulin, C peptide, glucagon, or insulin-like growthfactor I. IFN- did not alter plasma concentrations of glucose andfree fatty acids nor the rate of appearance of glucose. IFN-increased resting energy expenditure significantly. We conclude thatIFN- is a minor stimulator of the endocrine and metabolic pathways.Therefore, IFN- by itself is probably not a major mediator in theinteraction between the immune and the endocrine and metabolic systems.