Application of artificial gel antibodies for investigating molecular polymorphisms of human pituitary growth hormone.

Artificial gel antibodies were used to investigate human growth hormone (GH) activity in preparations purified from human pituitary glands. A partially purified fraction containing differently sized structural variants of GH was processed to yield monomeric and dimeric forms suitable for synthesizing artificial polyacrylamide gel antibodies. These two types of GH antibodies were used for investigating GH activity in experiments using HPLC gel-permeation and ion-exchange chromatography. In the size-exclusion experiments, both hormone fractions eluted as homogeneous peaks, whereas the ion exchanger resolved the hormones into several active components. The GH monomer antibodies exhibited a much higher affinity for monomeric GH than for dimeric GH, and the GH dimer antibodies exhibited a much higher affinity for dimeric GH than for monomeric GH. It was concluded that these two sets of antibodies might be useful for discriminating between dimeric and monomeric GH in clinical samples.

Acute 19-nortestosterone transiently suppresses hippocampal MAPK pathway and the phosphorylation of the NMDA receptor.

High doses of anabolic androgenic steroid are associated with changes in personality, e.g. increased aggression and irritability, behavioural changes that may be linked to structural changes in the hippocampus. In this in vivo study we demonstrate acute effects of a single injection of 19-nortestosterone on proteins that play a major role in molecular plasticity at synaptic connections. The steroid rapidly and transiently decreased total and phosphorylated NMDA receptor GluN2B subunit levels and phosphorylated extracellular signal-regulated kinase 1 in rat hippocampal synaptoneurosomes. Pretreatment with the androgen receptor antagonist flutamide prevented these effects suggesting an androgen receptor mediated mode of action. However, flutamide alone stimulated the phosphorylation of both extracellular signal-regulated kinase 1 and 2. EphrinB2 and phosphorylated translation initiation factor 4E, two proteins that act on synaptic plasticity through NMDA receptor and/or mitogen-activated protein kinase pathways, were not affected by any of the treatment regimens. This study demonstrates rapid in vivo effects of an anabolic androgenic steroid on two key elements in hippocampal synaptic plasticity.

Development and evaluation of normalization methods for label-free relative quantification of endogenous peptides.

The performances of 10 different normalization methods on data of endogenous brain peptides produced with label-free nano-LC-MS were evaluated. Data sets originating from three different species (mouse, rat, and Japanese quail), each consisting of 35-45 individual LC-MS analyses, were used in the study. Each sample set contained both technical and biological replicates, and the LC-MS analyses were performed in a randomized block fashion. Peptides in all three data sets were found to display LC-MS analysis order-dependent bias. Global normalization methods will only to some extent correct this type of bias. Only the novel normalization procedure RegrRun (linear regression followed by analysis order normalization) corrected for this type of bias. The RegrRun procedure performed the best of the normalization methods tested and decreased the median S.D. by 43% on average compared with raw data. This method also produced the smallest fraction of peptides with interblock differences while producing the largest fraction of differentially expressed peaks between treatment groups in all three data sets. Linear regression normalization (Regr) performed second best and decreased median S.D. by 38% on average compared with raw data. All other examined methods reduced median S.D. by 20-30% on average compared with raw data.

A quantitative peptidomic analysis of peptides related to the endogenous opioid and tachykinin systems in nucleus accumbens of rats following naloxone-precipitated morphine withdrawal.

We have applied a recently developed label-free mass spectrometry based peptidomic approach to identify and quantify a variety of endogenous peptides from rat nucleus accumbens following withdrawal in naloxone-precipitated, morphine-dependent rats of two separate strains. We focused on maturated, partially processed and truncated peptides derived from the peptide precursors proenkephalin, prodynorphin and preprotachykinin. The expression of several identified peptides was dependent on strain and was affected during morphine withdrawal.

Nandrolone-induced hippocampal phosphorylation of NMDA receptor subunits and ERKs.

The age-related decline in gonadal steroids is associated with changes in mood and memory function. It appears that normal physiological concentrations of the steroids are required for adequate synaptic plasticity. However, the effects of high levels of androgens subsequent to misuse of anabolic androgenic steroids (AAS) are largely unknown. In this study, rats were given i.m. nandrolone as a single dose or daily for 14 days and the effects on synaptic components in hippocampal synaptoneurosomes were measured 24h after the last injection. Western blot analysis revealed that a single injection of AAS increased phosphorylation of the NMDA receptor subunits NR2A and NR2B and ERK1/2, while the levels of phosphorylated CaMKIIalpha were unaltered. No changes were seen in other synaptic proteins tested, i.e., BDNF, Arc, TUC-4, and beta-tubulin III. Daily administration of nandrolone for 2 weeks did not affect the content of any of the proteins tested. From this in vivo study, it is concluded that important synaptic components respond to a single high dose of nandrolone, an effect that may influence synapse function.

Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A.

The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.