Genetic enhancement of behavioral itch responses in mice lacking phosphoinositide 3-kinase-γ (PI3Kγ)

Protein interacting with C Kinase 1 (PICK1), a PDZ domain-containing scaffolding protein, interacts with multiple different proteins in the mammalian nervous system and is believed to play important roles in diverse physiological and pathological conditions. In this study, we report that PICK1 is expressed in neurons of the dorsal root ganglion (DRG) and spinal cord dorsal horn, two major pain-related regions. PICK1 was present in approximately 29.7% of DRG neurons, most of which were small-less than 750 μm² in cross-sectional area. Some of these PICK1-positive cells co-labeled with isolectin B4 or calcitonin-gene-related peptide. In the dorsal horn, PICK1 immunoreactivity was concentrated in the superficial dorsal horn, where it was prominent in the postsynaptic density, axons, and dendrites. Targeted disruption of PICK1 gene did not affect basal paw withdrawal responses to acute noxious thermal and mechanical stimuli or locomotor reflex activity, but it completely blocked the induction of peripheral nerve injury-induced mechanical and thermal pain hypersensitivities. PICK1 appears to be required for peripheral nerve injury-induced neuropathic pain development and to be a potential biochemical target for treating this disorder.     

Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.     

A protocol for obtaining embryogenic cell lines from Arabidopsis

Embryogenic cell lines with lasting embryogenic potential can be obtained from somatic embryos induced directly from zygotic embryos of Arabidopsis thaliana , ecotype Columbia. The response to a critical concentration of auxin, which seems to be the all-important factor in the generation of embryogenic cell lines, is exhibited by somatic embryos but not by zygotic ones. The basis for this differential response remains obscure and will be discussed in relation to other systems.     

Decrease of Acetylcholine Release from Cortical Slices in Aged Rats: Investigations into Its Reversal by Phosphatidylserine

The release of total acetylcholine (ACh) and [3H]ACh was investigated in electrically stimulated cortical slices prepared from 4- and 18-month-old male Wistar rats. The slices were prelabeled with [3H]choline ([3H]Ch) and perfused with Krebs solution containing physostigmine. Total ACh was measured and the nature of the tritium efflux identified by HPLC. The total tritium content in the slices at the end of the incubation period was half as great in the old as in young rats. A linear relationship was found between stimulation frequencies (2, 5, and 10 Hz) and fractional [3H]ACh release in both young and old rats. In the latter the release was significantly smaller. At 10 Hz stimulation frequency the ratio between the two 2-min stimulation periods, S2/S1, was higher in the 18-month-old rats than in the young rats. Specific activity of the evoked ACh release was significantly smaller in S2 than in S1 in 4-month-old rats only. These findings indicate that the young synthetize ACh from endogenous unlabeled Ch more than older rats. In 18-month-old rats both the evoked total ACh and [3H]ACh release, expressed as picograms per minute, showed an approximately 50% decrease in both S1 and S2 stimulation periods, with no significant difference in specific activity. Phosphatidylserine (PtdSer) administration (15 mg/kg, i.p. daily) for 1 week to 18-month-old rats prevented the reduction in total evoked ACh release but not the reduction in evoked [3H]ACh release. The specific activity of ACh release was therefore significantly smaller than that of the young and untreated old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the low-molecular-mass (type X) collagen from chick-embryo tibial cartilage

Type X collagen, synthesized in large amount by cultured tibial chondrocytes, is deposited in vivo in the epiphyseal cartilages of 17-day-old chick embryo tibiae. Here we report the extraction of this collagen from these cartilages by limited pepsin digestion and its purification to electrophoretic homogeneity by salt precipitation followed by agarose gel filtration. Identity of the collagen purified from cartilage with the type X collagen synthesized by cultured chondrocytes is confirmed by comparison of the amino acid compositions. The high glycosylation extent of type X collagen is reminiscent of the glycosylation extent of pericellular collagens. The possible role of type X collagen is discussed.     

Regulation of the Na,K-ATPase activity of Madin-Darby canine kidney cells in defined medium by prostaglandin E1 and 8-bromocyclic AMP.

The role of PGE1 in regulating the activity of the Na+, K(+)-ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain-sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5-day period. The increase in the initial rate of ouabain-sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6-fold increase in the Vmax for ouabain-sensitive Rb+ uptake. The increase in the Vmax for ouabain-sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K(+)-ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K(+)-ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8-bromocyclic AMP treated MDCK monolayers, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8-bromocyclic AMP on the Vmax for ouabain-sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain-sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8-bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K(+)-ATPase activity in these variant cells.