Suppression of A-type potassium current in pyramidal neurons of the rat hippocampus, induced by pinacidil and its fluorine derivatives

With the use of a patch-clamp technique in the whole-cell configuration, we studied the effects of pinacidil and its fluorine derivatives on A-type potassium current (I _A) through the membrane of pyramidal neurons of the rat hippocampus. Hydrogen peroxide (10 mM) exerted no influence on the rate of inactivation ofI _A; therefore, this current is probably mediated by Shal Kv4.2 potassium channels. Pinacidil demonstrated the properties of a weakI _A blocker: in the 500 μM concentration it blocked about 45% of the current, while 50 μM of pinacidil fluorine derivatives were capable of blocking up to 30% ofI _A. The effects of pinacidil and its derivatives showed no dependence on the stimulating potential. A similar pattern of the effects of pinacidil fluorine derivatives, which are an order of magnitude stronger than those of pinacidil itself, allows us to suppose that the imine nitrogen of the tested compounds is significantly more involved in the molecular interaction with the site of an A-type potassium channel than the pyridine nitrogen.     

Impact of pre-culture on short- and long-term in vitro recovery of the biosynthetic potential and enzymatic and non-enzymatic antioxidant defense of Hypericum rumeliacum Boiss. after cryostorage

Due to its high hypericin and pseudohypericin in vitro biosynthetic capacity, the Balkan endemic Hypericum rumeliacum was selected as a prospective candidate for long-term preservation of valuable medicinal plant germplasm. Initial cryopreservation experiments were previously conducted based on the successful protocol established and reported for the widely studied H. perforatum. This is the first report on the impact of pre-culture duration on the short- and long-term in vitro recovery of the biosynthetic potential and antioxidant defense system of H. rumeliacum cryopreserved by vitrification. Cryopreservation did not impair the phenolics and flavonoids production of the regenerated plants. Moreover, hypericin and pseudohypericin levels even increased substantially in one of the regenerated lines, reaching yields from 0.107 and 0.752?mg?g^?1?DW in the control up to 0.277 and 1.112?mg?g^?1?DW for hypericin and pseudohypericin, respectively. However, the physical injury stress of the pre-culture treatment manipulations affected the physiological status of regenerants in a time dependent manner. Within 6?months after thawing, regenerants with the highest oxidative stress after pre-culture, were characterized with an augmentation of antioxidant metabolites such as phenolics, flavonoids, glutathione and ascorbic acid as well as increased antioxidant enzymatic activities in comparison with both the non-frozen control and the regenerants with the lowest pre-culture oxidative stress. Then, after 18?months of recovery, the same first H. rumeliacum group displayed a marked drop of enzymatic antioxidant activity as compared with the other groups of plants. Further research is needed to target oxidative stress alleviation to optimize H. rumeliacum cryopreservation protocol.     

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.     

Prdm9 and Meiotic Cohesin Proteins Cooperatively Promote DNA Double-Strand Break Formation in Mammalian Spermatocytes

1. Download high-res image (207KB)
2. Download full-size image

     

Insights into the evolution of the nucleolus by an analysis of its protein domain repertoire

Recently, the first investigation of nucleoli using mass spectrometry led to the identification of 271 proteins. This represents a rich resource for a comprehensive investigation of nucleolus evolution. We applied a protocol for the identification of known and novel conserved protein domains of the nucleolus, resulting in the identification of 115 known and 91 novel domain profiles. The phyletic distribution of nucleolar protein domains in a collection of complete proteomes of selected organisms from all domains of life confirms the archaebacterial origin of the core machinery for ribosome maturation and assembly, but also reveals substantial eubacterial and eukaryotic contributions to nucleolus evolution. We predict that, in different phases of nucleolus evolution, protein domains with different biochemical functions were recruited to the nucleolus. We suggest a model for the late and continuous evolution of the nucleolus in early eukaryotes and argue against an endosymbiotic origin of the nucleolus and the nucleus. Supplementary material for this article can be found on the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html.     

Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.     

Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs.     

Monosomy 11Q: report of new phenotypic manifestations.

We present a case of new phenotypic findings not previously reported associated with a partial deletion of chromosome 11 with a break point at 23q - (46,XY,del(11)(q23). Partial deletion of chromosome 11q was first described by Jacobsen et al(4). Forty-eight patients have been reported during the last 30 years, with variable break points between 11q11 and 11qter. New phenotypic findings in our patient with the associated 11q deletion are imperforate anus, bilateral cataracts, and hypoplastic, multilobed lungs.     

Crossover interference underlies sex differences in recombination rates

In many organisms, recombination rates differ between the two sexes. Here we show that in mice, this is because of a shorter genomic interference distance in females than in males, measured in Mb. However, the interference distance is the same in terms of bivalent length. We propose a model in which the interference distance in the two sexes reflects the compaction of chromosomes at the pachytene stage of meiosis.