Ancestral roles of the Fam20C family of secreted protein kinases revealed in C. elegans.

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the Caenorhabditis elegans Fam20C orthologue, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains intercellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in these contexts. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical, and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain

6-OHDA-induced dopaminergic neurodegeneration in <i>Caenorhabditis elegans</i> is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33

<div><p>Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinson’s disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting <i>Caenorhabditis elegans</i> dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the <u>t</u>rans<u>t</u>hyretin-<u>r</u>elated gene <i>ttr-33</i>. The only described <i>C</i>. <i>elegans</i> transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of <i>C</i>. <i>elegans</i> larvae and is predicted to be a secreted protein. TTR-33 protects <i>C</i>. <i>elegans</i> from oxidative stress induced by paraquat or H₂O₂ at an organismal level. The increased oxidative stress sensitivity of <i>ttr-33</i> mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the <i>C</i>. <i>elegans</i> cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.</p></div

Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting beta TC3-cells by enhancement of intracellular calcium mobilization.

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (&gt; or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action

Single-sweep spectral analysis of contact heat evoked potentials:A novel approach to identify altered cortical processing after morphine treatment

AIMS: The cortical response to nociceptive thermal stimuli recorded as contact heat evoked potentials (CHEPs) may be altered by morphine. However, previous studies have averaged CHEPs over multiple stimuli, which are confounded by jitter between sweeps. Thus, the aim was to assess single-sweep characteristics to identify alterations induced by morphine. METHODS: In a crossover study 15 single-sweep CHEPs were analyzed from 62 electroencephalography electrodes in 26 healthy volunteers before and after administration of morphine or placebo. Each sweep was decomposed by a continuous wavelet transform to obtain normalized spectral indices in the delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), beta (12–32 Hz) and gamma (32–80 Hz) bands. The average distribution over all sweeps and channels was calculated for the four recordings for each volunteer, and the two recordings before treatments were assessed for reproducibility. Baseline corrected spectral indices after morphine and placebo treatments were compared to identify alterations induced by morphine. RESULTS: Reproducibility between baseline CHEPs was demonstrated. As compared with placebo, morphine decreased the spectral indices in the delta and theta bands by 13% (P = 0.04) and 9% (P = 0.007), while the beta and gamma bands were increased by 10% (P = 0.006) and 24% (P = 0.04). CONCLUSION: The decreases in the delta and theta band are suggested to represent a decrease in the pain specific morphology of the CHEPs, which indicates a diminished pain response after morphine administration. Hence, assessment of spectral indices in single-sweep CHEPs can be used to study cortical mechanisms induced by morphine treatment

Single-copy insertion of transgenes in Caenorhabditis elegans.

Currently transgenes in C. elegans are generated by injecting DNA into the germline. The DNA assembles into a semi-stable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method called MosSCI (Mos1-mediated Single Copy Insertion) that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double strand break in non-coding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single copy insertions can be obtained in less than two weeks by injecting approximately twenty animals. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines