No abstract

Intracellular delivery of bioactive molecules using arginine-rich peptides, including oligoarginine and HIV-1 Tat peptides, is a recently developed technology. Here, we report a dramatic change in the methods of internalization for these peptides brought about by the presence of pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. In the absence of pyrenebutyrate, endocytosis plays a major role in cellular uptake. However, the addition of pyrenebutyrate results in direct membrane translocation of the peptides yielding diffuse cytosolic peptide distribution within a few minutes. Using this method, rapid and efficient cytosolic delivery of the enhanced green fluorescent protein (EGFP) was achieved in cells including rat hippocampal primary cultured neurons. Enhancement of bioactivity on the administration of anapoptosis-inducing peptide is also demonstrated. Thus, coupling arginine-rich peptides with this hydrophobic anion dramatically improved their ability to translocate cellular membranes, suggesting the great impact of this approach on exploring and controlling cell function.

We have identified a novel glutamate receptor subunit on the human and mouse genome. Cloning of the mouse cDNA revealed a protein consisting of 1003 amino acids encoded by at least nine exons. This protein showed the highest similarity (51%) to the NR3A subunit of the NMDA receptor and therefore was termed NR3B. NR3B has a structure typical of glutamate receptor family members with a signal peptide and four membrane-associated regions. Amino acids forming a ligand-binding pocket are conserved. When coexpressed with NR1 and NR2A in heterologous cells, NR3B suppressed glutamate-induced current similarly to NR3A. Thus members of the NR3 class of NMDA receptors act as dominant-negative subunits in the NMDA receptor complex. NR3B shows very restricted expression in somatic motoneurons of the brainstem and spinal cord. Its expression in other types of motoneurons, including autonomic motoneurons in Onuf's nucleus and oculomotor neurons, is significantly weaker. Our results indicate that NR3B is important as a regulatory subunit that controls NMDA receptor transmission in motoneurons. It may be involved in the pathogenesis of neurodegenerative diseases involving motoneurons as well.

Neurons in the upper lumbar spinal cord project axons containing gastrin-releasing peptide (GRP) to innervate lower lumbar regions controlling erection and ejaculation. This system is vestigial in female rats and in males with genetic dysfunction of androgen receptors, but in male rats, pharmacological stimulation of spinal GRP receptors restores penile reflexes and ejaculation after castration. GRP offers new avenues for understanding potential therapeutic approaches to masculine reproductive dysfunction.GRP, a member of the bombesin-like peptide family 1 , is distributed widely in the central nervous system and gastrointestinal tract of mammals 2 , 3 . GRP and neuromedin B (NMB), the mammalian counterpart of bombesin, play a role in many physiological processes, including itch 4 , circadian rhythms 5 , food intake 6 and fear memory consolidation 7 , 8 . In mammals, bombesin-like peptides act through a family of at least three G protein-coupled receptors: GRP-preferring receptor (GRP-R), NMB-preferring receptor (NMB-R) and bombesin receptor subtype-3 (BRS-3) 9 .Using immunocytochemistry (ICC) directed at GRP, we found a group of neurons within a region previously dubbed the 'spinal ejaculation generator' because toxins that selectively lesion galanin-containing neurons there virtually eliminate ejaculation in rats 10 . These galaninergic neurons project to the thalamus 10 , but it had been unclear whether there are also direct connections between this center and the lower spinal cord regions that directly trigger ejaculation 11 . The separate, GRP-containing neurons that we found within the center projected axons to more caudal spinal regions and were much more prominent in wild-type (WT) males than in WT females ( Fig. 1a,b; Supplementary Fig. 1 online) (n = 5, F 2,12 = 299.9, P < 0.001). Semiquantitative reverse transcription (RT)-PCR confirmed more pre-pro NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptGrp transcripts in this region of males than of females ( Supplementary Fig. 2 online). To test whether androgen receptors direct sexual dimorphism of these neurons, we examined genetically male (XY) Long-Evans rats carrying the testicular feminization mutation (Tfm) of the androgen receptor gene Ar. These rats develop testes embryologically and secrete testosterone pre-and postnatally but, because their androgen receptor protein is dysfunctional, develop a wholly feminine exterior, including a clitoris rather than a penis. The spinal cord of Tfm rats was hyperfeminine, having even fewer GRP-positive neurons in this region than did WT females (P <0.001) (Fig. 1c,d). In normal males, GRP-expressing neurons also expressed androgen receptor (96.1 ± 1.7%; n = 4 WT males), but not estrogen receptor alpha (ERα) (Fig. 1e-h). Because androgens such as testosterone augment ejaculation in male rats and humans 12 , the presence of androgen receptor in the GRPpositive neurons of the ejaculation center offers a locus for androgenic modulation of ejaculation and other sexual reflexes.ICC r...

Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to be increased in liver fibrosis development both in murine experimental models and human samples. However, the direct role of TIMP-1 during liver fibrosis development has not been defined. To address this issue, we developed transgenic mice overexpressing human TIMP-1 (hTIMP-1) in the liver under control of the albumin promoter/ enhancer. A model of CCl(4)-induced hepatic fibrosis was used to assess the extent of fibrosis development in TIMP-1 transgenic (TIMP-Tg) mice and control hybrid (Cont) mice. Without any treatment, overexpression of TIMP-1 itself did not induce liver fibrosis. There were no significant differences of pro-(alpha1)-collagen-I, (alpha2)-collagen-IV, and alpha-smooth muscle actin (alpha-SMA) mRNA expression in the liver between TIMP-Tg and Cont-mice, suggesting that overexpression of TIMP-1 itself did not cause hepatic stellate cell (HSC) activation. After 4-week treatment with CCl(4), however, densitometric analysis revealed that TIMP-Tg-mice had a seven-fold increase in liver fibrosis compared with the Cont-mice. The hepatic hydroxyproline content and serum hyaluronic acid were also significantly increased in TIMP-Tg-mice, whereas CCl(4)-induced liver dysfunction was not altered. An active form of matrix metalloproteinases-2 (MMP-2) level in the liver of TIMP-Tg-mice was decreased relative to that in Cont-mice because of the transgenic TIMP-1. Immunohistochemical analysis revealed that collagen-I and collagen-IV accumulation was markedly increased in the liver of CCl(4)-treated TIMP-Tg-mice with a pattern similar to that of alpha-SMA positive cells. These results suggest that TIMP-1 does not by itself result in liver fibrosis, but strongly promotes liver fibrosis development.