Differential enantioselective effects of pentyl-4-yn-valproate on spatial learning in the rat, and neurite outgrowth and cyclin D3 expression in vitro

Previously, we demonstrated the racemic form of the valproate (VPA) analogue, 2-n-pentyl-4-pentynoic acid ([+/-]pentyl-4-yn-VPA), to be neuritogenic in vitro and to enhance cognition in vivo. To determine the enantioselectivity of these effects, the racemate and purified enantiomers of [+/-]pentyl-4-yn-VPA (84 mg/kg, i.p.) were administered to rodents 20 min prior to multi-session water maze training. The racemate and R-enantiomer significantly reduced escape latencies during water maze learning and enhanced its recall in a probe trial 3 days later. In contrast, S-pentyl-4-yn-VPA did not influence these behavioural parameters. The enantiomer-specific effects of [+/-]pentyl-4-yn-VPA were further discriminated in vitro using neuro 2A neuroblastoma and C6 glioma cell lines. In neuro 2A, the S-enantiomer induced profound neurite outgrowth at concentrations up to 0.5 mm, with the R-enantiomer and racemate being less neuritogenic. Immunoblot analysis of cyclin D3 expression in C6 glioma indicated the racemate and S-pentyl-4-yn-VPA to induce dose-dependent up-regulation of this protein, similar to that associated with G1-phase cell cycle arrest mediated by VPA, whereas R-pentyl-4-yn-VPA was without effect. These results indicate that the cognition-enhancing effects of pentyl-4-yn-VPA are due to the actions of the R-enantiomer, and that cyclin D3 up-regulation and associated anti-proliferative and pro-differentiative actions are predominantly associated with the S-enantiomer.     

Solid-state NMR spin diffusion for measurement of membrane-bound peptide structure: gramicidin A

A recently developed solid-state NMR method for measurement of depths in membrane systems is applied to gramicidin A, a membrane-bound peptide of known structure, to investigate the potential of this method. (15)N-detected, (1)H spin diffusion experiments demonstrate the resolution of the technique by measuring the 4-5 A depth differences between three (15)N-labeled backbone sites (Trp13, Val7, Gly2) in gramicidin A. We also show that (13)C-detected, (1)H spin diffusion experiments on unlabeled gramicidin A are sufficient to discriminate between the end-to-end dimer and double-helix structures of gramicidin A. Thus, spin diffusion solid-state NMR experiments can provide a simple approach, which does not require labeled samples, for testing structural models of membrane-bound peptides.     

Comparative cytogenetics of the African elephant (Loxodonta africana) and Asiatic elephant (Elephas maximus)

G- and C-banded karyotypes of the two extant species of the mammalian order Proboscidea are presented for the first time. Chromosome complements were 2n = 56 in both Loxodonta africana and Elephas maximus. Comparisons between the species demonstrated a high level of chromosome band homology, with 26 conserved autosomal pairs. The normal diploid karyotype of L. africana had 25 acrocentric/telocentric and two metacentric/submetacentric autosomal pairs. E. maximus differed by having one less acrocentric and one additional submetacentric pair due to either a heterochromatic arm addition or deletion involving autosomal pair 27. Several acrocentric autosomes of L. africana exhibited small short arms that were absent in homologous chromosomes of E. maximus. The X chromosomes in both species were large submetacentric elements and were homologous. However, the small acrocentric Y chromosomes differed; in E. maximus it was slightly larger and had more distinct G-bands than its counterpart in L. africana. Extant Elephantidae appear to be relatively conservative in their rates of chromosomal change compared to some other mammalian families. The high-quality banded karyotypes presented here should prove useful as references in future chromosome analyses of elephant populations and in comparative cytogenetic studies with other ungulate orders.     

Calmodulin remains extended upon binding to smooth muscle caldesmon: a combined small-angle scattering and fourier transform infrared spectroscopy study

We show that calmodulin (CaM) has an extended conformation in its complexes with sequences from the smooth muscle thin filament protein caldesmon (CaD) by using small-angle X-ray and neutron scattering with contrast variation. The CaD sequences used in these experiments were a C-terminal fragment, 22kCaD, and a smaller peptide sequence within this fragment, MG56C. Each of these sequences contains the CaM-binding sites A and B previously shown to interact with the C- and N-terminal lobes of CaM, respectively [Wang et al. (1997) Biochemistry 36, 15026]. By modeling the scattering data, we show that the majority of the MG56C sequence binds to the N-terminal domain of CaM. FTIR data on CaM complexed with 22kCaD or with MG56C peptide show the 22kCaD sequence contains unordered, helix, and extended structures, and that the extended structures reside primarily in the MG56C portion of the sequence. There are small changes in secondary structure, involving approximately 12 residues, induced by CaM binding to CaD. These changes involve a net decrease in extended structures accompanied by an increase in alpha-helix, and they occur within the CaM and/or in the MG56C sequence.     

Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2

The interaction of heparan sulfate (HS) (and the closely related molecule heparin) with FGF-1 is a requirement for enabling the growth factor to activate its cell surface tyrosine kinase receptor. However, little is known about the regulatory role of naturally occurring cell surface HS in FGF-1 activation. We have addressed this issue by utilizing a library of HS oligosaccharides, which are defined in both length and sulfate content. Mitogenic activation assays using these oligosaccharides showed that HS contained both FGF-1 activatory and inhibitory sugar sequences. Further analysis of these oligosaccharides showed a clear correlation between FGF-1 promoting activity and their 6-O-sulfate content. The results, in particular with the dodecasaccharide sequences, suggested that specific positioning of 6-O-sulfate groups may be required for the promotion of FGF-1 mitogenic activity. This may also be true for 2-O-sulfate groups though the evidence was not as conclusive. Differential activation of FGF-1 and FGF-2 was also observed and found to be mediated by both oligosaccharide length and sulfation pattern, with different specific O-sulfate positioning being implicated for the promotion of different growth factors. These results suggest that variation and tight control of the fine structure of HS may allow cells to not only control their positive/negative responses to individual FGFs but also to change specificity towards promotion of different members of the FGF family.     

Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels

A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.     

NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox^-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox^-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox^-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox^-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox^-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction.     

Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable

Many of the biological functions attributed to cell surface heparan sulfate (HS) proteoglycans, including the Syndecan family, are elicited through the interaction of their HS chains with soluble extracellular molecules. Tightly controlled, cell-specific sulfation and epimerization of HS precursors endows these chains with highly sulfated, iduronate-rich regions, which are major determinants of cytokine and matrix-protein binding and which are interspersed by N-acetylated, poorly sulfated regions. Until this study, there have been no comprehensive structural comparisons made on HS chains decorating simultaneously expressed, but different, syndecan core proteins. In this paper we demonstrate that the HS chains on affinity-purified syndecan-1 and -4 from murine mammary gland cells are essentially identical by a number of parameters. Size determination, disaccharide analyses, enzymatic and chemical scission methods, and affinity co-electrophoresis all failed to reveal any significant differences in fine structure, domain organization, or ligand-binding properties of these HS species. These findings lead us to suggest that the imposition of the fine structure onto HS occurs independently of the core protein to which it is attached and that these core proteins, in addition to the HS chains, may play a pivotal role in the various biological functions ascribed to these macromolecules.