Selective activation of classical and alternative pathways of human complement by "promptly serum-sensitive" and "delayed serum-sensitive" strains of Serratia marcescens.

Chelation of fresh human serum with 0.01 M MgCl2 (Mg) plus 0.01 M ethylene glycol tetraacetic acid failed to abrogate the bactericidal activity against "delayed serum-sensitive" strains of Serratia marcescens, whereas previously "promptly serum-sensitive" strains of S. marcescens and control strain Escherichia coli C were killed after an extended period of incubation. The addition of 0.01 M ethylenediametetracetate to fresh human serum neutralized bactericidal activity against S. marcescens of either serum sensitivity category.     

Mutations in the human melanocortin-4 receptor gene associated with severe familial obesity disrupts receptor function through multiple molecular mechanisms

Mutations in the melanocortin-4 receptor gene (MC4R) represent the commonest monogenic cause of human obesity. However, information regarding the precise effects of such mutations on receptor function is very limited. We examined the functional properties of 12 different mutations in human MC4R that result in severe, familial, early-onset obesity. Of the nine missense mutants studied, four were completely unable to generate cAMP in response to ligand and five were partially impaired. Four showed evidence of impaired cell surface expression and six of reduced binding affinity for ligand. One mutation in the C-terminal tail, I316S, showed reduced affinity for alpha-MSH but retained normal affinity for the antagonist AgRP. None of the mutations inhibited signaling through co-transfected wild-type receptors. Thus, in the most comprehensive study to date of the functional properties of naturally occurring MC4R mutations we have (1) established that defective expression on the cell surface is a common mechanism impairing receptor function, (2) identified mutations which specifically affect ligand binding affinity thus aiding the definition of receptor structure-function relationships, (3) provided evidence against the notion that these receptor mutants act as dominant-negatives, and (4) identified a potentially novel molecular mechanism of receptor dysfunction whereby a mutation alters the relative affinities of a receptor for its natural agonist versus antagonist.     

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.     

A de novo mutation affecting human TrkB associated with severe obesity and developmental delay

An 8-year-old male with a complex developmental syndrome and severe obesity was heterozygous for a de novo missense mutation resulting in a Y722C substitution in the neurotrophin receptor TrkB. This mutation markedly impaired receptor autophosphorylation and signaling to MAP kinase. Mutation of NTRK2, which encodes TrkB, seems to result in a unique human syndrome of hyperphagic obesity. The associated impairment in memory, learning and nociception seen in the proband reflects the crucial role of TrkB in the human nervous system.     

Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse

Summary:  Natural killer (NK) cell cytotoxicity is mediated by multiple germ line-encoded activating receptors that recognize specific ligands expressed by tumor cells and virally infected cells. These activating receptors are opposed by NK inhibitory receptors, which recognize major histocompatibility complex class I molecules on potential targets, raising the threshold for NK cell activation. Once an abnormal cell has been detected, NK cells are the sentinel source of cytolytic mediators, such as granzymes and perforins, as well as interferon-γ, which can polarize the immune response to a T-helper 1 cell type. Activation signals are transmitted by adhesion-dependent pathways, immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathways, DAP10 ITAM-independent pathways, and by signaling through immunoreceptor tyrosine-based switch motifs. These pathways activate downstream signaling partners to trigger NK cell cytotoxicity. Some of these downstream molecules are unique to the various pathways, and some of these molecules are shared. Because of the complexity of signals involved in NK cell–target cell interaction, the generation of mice with targeted mutations in signaling molecules involved in adhesion, activation, or inhibition is essential for a precise dissection of the mechanisms regulating NK cell effector functions. Here we review recent advances in the genetic analysis of the signaling pathways that mediate NK cell killing.     

Fast network oscillations in the rat dentate gyrus in vitro

The dentate gyrus is a prominent source of gamma frequency activity in the hippocampal formation in vivo. Here we show that transient epochs of gamma frequency network activity (67 +/- 12 Hz) can be generated in the dentate gyrus of rat hippocampal slices, following brief pressure ejections of a high-molarity potassium solution onto the molecular layer. Oscillatory activity remains synchronized over distances >300 microm and is accompanied by a modest rise in [K(+)](o). Gamma frequency oscillations were abolished by a GABA(A) receptor antagonist demonstrating their dependence on rhythmic inhibition. However, in many cases, higher frequency oscillations (>80 Hz) remained in the absence of synaptic transmission, thus demonstrating that nonsynaptic factors may underlie fast oscillatory activity.     

Humoral immune response to thymidylate synthase in colon cancer patients after 5-FU chemotherapy

Thymidylate synthase (TYMS), the critical enzyme for DNA synthesis and a target for chemotherapy, was recently characterized as an oncogene and a potential target for specific immunotherapy. Here we report TYMS-specific antibody response in a fraction of colon cancer patients. Humoral immune response to TYMS is induced by chemotherapy using TYMS inhibitors, such as 5-fluorouracil (5-FU), and may be associated with tumor burden. Therefore, TYMS may serve as a useful serological biomarker for monitoring the course of disease and treatment in cancer patients.