GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Superoxide dismutase,catalase, and U78517F attenuate neuronal damage in gerbils with repeated brief ischemic insults

Repeated ischemic insults at one hour intervals result in more severe neuronal damage than a single similar duration insult. The mechanism for the more severe damage with repetitive ischemia is not fully understood. We hypothesized that the prolonged reperfusion periods between the relatively short ischemic insults may result in a pronounced generation of oxygen free radicals (OFRs). In this study, we tested the protective effects of superoxide dismutase (SOD) and catalase (alone or in combination), and U78517F in a gerbil model of repetitive ischemia. Three episodes (two min each) of bilateral carotid occlusion were used at one hour intervals to produce repetitive ischemia. Superoxide dismutase and catalase were infused via osmotic pumps into the lateral ventricles. Two doses of U78517F were given three times per animal, one half hour prior to each occlusion. Neuronal damage was assessed 7 days later in several brain regions using the silver staining technique. The Mann-Whitney U test was used for statistical comparison. Superoxide dismutase showed significant protection in the hippocampus (CA4), striatum, thalamus and the medial geniculate nucleus (MGN). Catalase showed significant protection in the striatum, hippocampus, thalamus, and MGN and the substantia nigra reticulata. Combination of the two resulted in additional protection in the cerebral cortex. Compared to the controls, there was little protection with a dose of 3 mg/kg of U78517F. There was significant protection with a dose of 10 mg/kg in the hippocampus (CA4), striatum, thalamus, medial geniculate nucleus and the substantia nigra reticulata. The significant protection noted with SOD, catalase or U78517F with repeated ischemia supports, the hypothesis that OFRs may play a role in neuronal damage in repeated cerebral ischemia.     

Human RAP1 inhibits non‐homologous end joining at telomeres

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, promote genome stability by distinguishing chromosome termini from DNA double‐strand breaks (DSBs). Cells possess two principal pathways for DSB repair: homologous recombination and non‐homologous end joining (NHEJ). Several studies have implicated TRF2 in the protection of telomeres from NHEJ, but the underlying mechanism remains poorly understood. Here, we show that TRF2 inhibits NHEJ, in part, by recruiting human RAP1 to telomeres. Heterologous targeting of hRAP1 to telomeric DNA was sufficient to bypass the need for TRF2 in protecting telomeric DNA from NHEJ in vitro. On expanding these studies in cells, we find that recruitment of hRAP1 to telomeres prevents chromosome fusions caused by the loss of TRF2/hRAP1 from chromosome ends despite activation of a DNA damage response. These results provide the first evidence that hRAP1 inhibits NHEJ at mammalian telomeres and identify hRAP1 as a mediator of genome stability.     

Pheromone peptide cOB1 from native Enterococcus faecalis forms amyloid‐like structures: A new paradigm for peptide pheromones

Pheromone peptides are an important component of bacterial quorum‐sensing system. The pheromone peptide cOB1 (VAVLVLGA) of native commensal Enterococcus faecalis has also been identified as an antimicrobial peptide (AMP) and reported to kill the prototype clinical isolate strain of E. faecalis V583. In this study, the pheromone peptide cOB1 has shown to form amyloid‐like structures, a characteristic which is never reported for a pheromone peptide so far. With in silico analysis, the peptide was predicted to be highly amyloidogenic. Further, under experimental conditions, cOB1 formed aggregates displaying characteristics of amyloid structures such as bathochromic shift in Congo red absorbance, enhancement in thioflavin T fluorescence, and fibrillar morphology under transmission electron microscopy. This novel property of pheromone peptide cOB1 may have some direct effects on the binding of the pheromone to the receptor cells and subsequent conjugative transfer, making this observation more important for the therapeutics, dealing with the generation of virulent and multidrug‐resistant pathogenic strains.     

Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11β-HSD1 inhibitors

Discovery and optimization of a piperidyl benzamide series of 11β-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.     

THE CONSEQUENCES OF BEING DIFFERENT: SINISTRAL COILING IN CERION

The overwhelming predominance of dextral coiling in gastropods is an outstanding and puzzling phenomenon. A few sinistral specimens (left coiling individuals) have been found in many dextral species. Only six sinistral shells have ever been found in Cerion; we base this analysis on the five available shells. We ask whether reversed symmetry is a simple either-or switch without further consequences for shell form, or whether sinistrality engenders associated effects, making left-coiling shells unlike their dextral deme-mates in other ways. All five sinistral shells differ in features of size and coiling late in growth, leading to relatively small apertures and a slight twist in the axis of coiling. We detect and measure this effect as follows: in multivariate morphospace, sinistrals occupy peripheral positions among their dextral deme-mates; in univariate analysis, sinistrals are consistently different for a set of characters involving covariance patterns never before seen in a decade of studies on ontogenetic and age-standardized variation in dextrals; a bootstrap procedure does not recover similar patterns in randomly constituted samples of dextrals matching the true sinistral distribution; direct x-ray measures of the coiling axis detect its slight twist in sinistrals. We discuss the implications of these unsuspected associations for the issues of developmental constraint upon the evolution of morphology.     

Multiple luteinizing hormone receptor (LHR) protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer's disease

Distinct luteinizing hormone receptor (LHR) protein variants exist due to the posttranslational modifications. Besides ovaries, LHR immunoreactivity (LHRI) was also found in other tissues, such as the brain, fallopian tube, endometrium, trophoblast and resident tissue macrophages. The 3B5 mouse monoclonal antibody was raised against purified rat LHR. In rat, porcine and human ovaries, the 3B5 identified six distinct LHR bands migrating at ~92, 80, 68, 59, 52 and 48 kDa. Characteristic LHRI was detected in rat, human and porcine corpora lutea. During cellular differentiation, subcellular LHR distribution changed from none to granular cytoplasmic, perinuclear, surface, nuclear and no staining. There were also differences in vascular LHR expression – lack of LHRI in ovarian vessels and strong staining of vessels in other tissues investigated. In normal human term placentae, villous LHRI was associated with blood sinusoids and cytotrophoblast cells, and rarely detected in trophoblastic syncytium. In all abnormal placentae, the LHRI of sinusoids was absent, and syncytium showed either enhanced (immature placental phenotypes) or no LHRI (aged placental phenotype). LHRI in human brain was identified in microglial cells (CD68+ resident macrophages). Protein extracts from human vaginal wall and levator ani muscle and fascia showed strong ~92 and 68 kDa species, and LHRI was detected in smooth muscle cells, fibroblasts, resident macrophages and nuclei of skeletal muscle fibers. Our observations indicate that, in contrast to the theory on the role of vascular hormone receptors in preferential pick up of circulating hormones, there is no need to enhance selective pick up rather only prevent LH/CG transport to inappropriate sites. Abnormal placental LHR expression may play a role in the development of abnormal pregnancy. Expression of LHR in the pelvic floor compartments suggests that high LH levels in postmenopausal women may contribute to the pelvic floor relaxation and increased incidence of pelvic floor disorders. Since chorionic gonadotropin increases secretion of a variety of cytokines by monocytes, and induces their inflammatory reaction and phagocytic activity, high LH levels in aging individuals may also activate microglia (mononuclear phagocyte system in the central nervous system) and contribute to the development of Alzheimer's disease and other inflammation-mediated neurodegenerative diseases.     

The effect of chronic fatty acid treatment on lipolysis in 3T3-L1 adipocytes.

Various saturated and unsaturated fatty acids were included in the culture medium to test their effects on lipolysis in 3T3-L1 adipocytes. Following prolonged incubation, only oleate was found to exert enhancing effect on basal and isoproterenol-stimulated lipolysis. The effect of oleate was concentration-dependent and was accompanied with increased intracellular cAMP content. Furthermore, the lipolytic response induced by isobutyl-methylxanthine, forskolin or dibutyryl cAMP was also increased in adipocytes treated with oleate. Thus, it appears that in addition to an increased cAMP accumulation, a step distal to cAMP production in the cells may be involved in inducing enhanced lipolysis in 3T3-L1 adipocytes by prolonged exposure to oleate.     

Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis

The generation of extracellular oxidants by neutrophils has been widely investigated, but knowledge about the chemical reactions that occur in the phagolysosome, the cellular compartment that kills pathogens, is more limited. One important pathway may involve the production of potent halogenating agents such as hypochlorous acid (HOCl) by the myeloperoxidase-hydrogen peroxide-halide system. However, explorations of the oxidation chemistry of phagolysosomes have been hampered by the organelle's inaccessibility. To overcome this limitation, we recovered Escherichia coli that had been internalized by human neutrophils. We then analyzed the bacterial proteins for 3-chlorotyrosine, a stable marker of damage by HOCl. Mass spectrometric analysis revealed that levels of 3-chlorotyrosine in E. coli proteins increased markedly after the bacteria were internalized by human neutrophils. This increase failed to occur in E. coli exposed to neutrophils deficient in NADPH oxidase or myeloperoxidase, implicating H(2)O(2) and myeloperoxidase in the halogenation reaction. The extent of protein chlorination by normal neutrophils paralleled bacterial killing. Our observations support the view that the phagolysosome of human neutrophils uses the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate bacterial proteins. In striking contrast, human neutrophils failed to nitrate bacterial proteins unless the medium was supplemented with 1 mm nitrite, and the level of nitration was low. Protein chlorination associated with bacterial killing was unaffected by the presence of nitrite in the medium. Nitration required NADPH oxidase but appeared to be independent of myeloperoxidase, suggesting that neutrophils can nitrate proteins through a pathway that requires nitrite but is independent of myeloperoxidase.     

Structural attributes of the hypogeous holoparasite Hydnora triceps Drege & Meyer (Hydnoraceae)

The morphology of the hypogeous root holoparasite Hydnora triceps is highly reduced, and as with many holoparasites, the vegetative body is difficult to interpret. The vegetative body of H. triceps has been historically considered a "pilot root" studded with lateral appendages known as "haustorial roots." We found the vegetative body of H. triceps to consist of a rhizome with a thickened root-cap-like structure that covered a vegetative shoot apical meristem. From the apical meristem, procambial strands originated and developed into endarch collateral vascular bundles arranged radially around a pith without an interfascicular cambium. Xylem vessels had scalariform pitting and simple perforation plates. A continuous periderm without root hairs was observed. Increase in girth was attributed to cork and fascicular cambia. "Haustorial roots" or bumps on the surface of the vegetative body were exogenous, contained meristems and were the origins of vegetative branching, budding, and haustoria. The haustoria of H. triceps were cylindrical and penetrated the host root stele. Phloem and xylem elements were observed within the endophyte, and direct xylem to host-xylem contacts were observed. The arrangement of vascular tissues and xylem anatomy of H. triceps are likely plesiomorphic features in light of Hydnoraceae's placement in the Piperales.