Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine

Aging and the progression of certain degenerative diseases are accompanied by increases in intracellular fluorescent material, termed "lipofuscin" and ceroid, respectively. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However, little direct evidence currently exists because the structures responsible for the fluorescent, cross-linked nature of this material are not well characterized. In this study, we have identified a fluorescent product formed in the reaction of Nalpha-acetyllysine and 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions [Esterbauer, H., Shaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11, 81-128]. This fluorescent compound, characterized as a 2-hydroxy-3-imino-1,2-dihydropyrrol derivative, appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link. Polyclonal antibody was raised to the Nalpha-acetyllysine-HNE fluorophore and found to be highly specific to the chromophore structure of the compound. This antibody has been used to conclusively demonstrate that the lysine-HNE derivative of this fluorophore forms on protein upon exposure to HNE. The results of this study therefore provide the basis for future investigations on the contribution(s) of HNE-derived fluorophore formation to lipofuscin and ceroid accumulation.     

Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis

The mechanism by which glucose stimulates insulin secretion from the pancreatic islets of Langerhans is incompletely understood. It has been suggested that malonyl-CoA plays a regulatory role by inhibiting fatty acid oxidation and promoting accumulation of cytosolic long-chain acyl-CoA (LC-CoA). In the current study, we have re-evaluated this "long-chain acyl-CoA hypothesis" by using molecular and pharmacologic methods to perturb lipid metabolism in INS-1 insulinoma cells or rat islets during glucose stimulation. First, we constructed a recombinant adenovirus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), an enzyme that decarboxylates malonyl-CoA to acetyl-CoA. INS-1 cells treated with AdCMV-MCD had dramatically lowered intracellular malonyl CoA levels compared with AdCMV-betaGal-treated cells at both 3 and 20 mM glucose. Further, at 20 mM glucose, AdCMV-MCD-treated cells were less effective at suppressing [1-14C]palmitate oxidation and incorporated 43% less labeled palmitate and 50% less labeled glucose into cellular lipids than either AdCMV-betaGAL-treated or untreated INS-1 cells. Despite the large metabolic changes caused by expression of MCD, insulin secretion in response to glucose was unaltered relative to controls. The alternative, pharmacologic approach for perturbing lipid metabolism was to use triacsin C to inhibit long-chain acyl-CoA synthetase. This agent caused potent attenuation of palmitate oxidation and glucose or palmitate incorporation into cellular lipids and also caused a 47% decrease in total LC-CoA. Despite this, the drug had no effect on glucose-stimulated insulin secretion in islets or INS-1 cells. We conclude that significant disruption of the link between glucose and lipid metabolism does not impair glucose-stimulated insulin secretion in pancreatic islets or INS-1 cells.     

Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2 (Th2) responses and require obligatory cytokine signals for induction of Th1 immunity

Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the "default" T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2-3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-gamma by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing- associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor alpha or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.     

A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000, forms complexes which are concentrated in the reticular laminae of electric organ basement membranes

Previously, we identified PG-1000 as part of a disulfide-linked complex of two large proteoglycans (PG-1000 and the beta component) and three smaller proteins purified from the extracellular matrix of elasmobranch electric organ (Iwata and Carlson, 1991, J. Biol. Chem. 266: 323-333). PG-1000 is a chondroitin sulfate/keratan sulfate proteoglycan with a molecular mass of about 1.2 x 16(6) daltons. When visualized in the electron microscope, PG-1000 has the typical "bottle-brush" appearance expected for a proteoglycan with an average total length of about 345 nm and about 20 chains of approximately 110 nm (Carlson and Wight, 1987, J. Cell Biol. 105: 3075-3086). Using immunocytochemical methods, we now demonstrate that PG-1000 is a component of the interstitial extracellular matrix of the electric organ. PG-1000 immunoreactivity is found throughout the interstitial matrix, but it is highly concentrated in that region of the matrix immediately adjacent to the basal lamina, the reticular lamina. The reticular and basal laminae together form the basement membrane. PG-1000 immunoreactivity is especially apparent on basal laminae that surround nerve fibers and nerve terminals. When the disulfide-linked PG-1000 complexes are purified and examined in the electron microscope following rotary shadowing, they appear as bottle-brush structures which are often attached at a central region and radiate like spokes of a wheel. These aggregates contain two to six proteoglycan monomers. We hypothesize that the PG-1000 complexes are disulfide-stabilized parts of an extended network of linked proteoglycans in the reticular lamina.     

Amblyomma variegatum (Acari: Ixodidae): mechanism and control of arbovirus secretion in tick saliva

Saliva is considered to be the conduit by which pathogens are transmitted from blood-sucking arthropod vectors to their vertebrate hosts, but supporting evidence for this is fragmentary. To determine if Thogoto (THO) virus, a tick-borne member of the influenza virus family, is transmitted via tick saliva, and whether virus replication is a prerequisite for such transmission, two experimental conditions were compared: (1) "biological transmission" and (2) "mechanical transmission." In (1), THO virus was allowed to infect and replicate in a natural vector, Amblyomma variegatum: virus was detected in saliva collected from 3/22 (14%) ticks. In (2), virus was inoculated directly into the hemocoel with the drug used to induce salivation and saliva was collected immediately to preclude the possibility of virus replication: virus was detected in saliva collected from 31/170 (18%) ticks. The results demonstrate that THO virus is secreted in tick saliva and that virus can pass from the hemolymph to the salivary glands independently of viral replication within the tick. The comparatively low numbers of ticks that yielded virus-positive saliva samples together with the results from assays of serial saliva samples suggested that virus secretion may not be a continuous process during salivation. Ticks in which THO virus had established an infection showed an impaired secretory response compared with uninfected ticks and ticks used for mechanical transmission.     

Video-assisted lateral intertransverse process arthrodesis. Validation of a new minimally invasive lumbar spinal fusion technique in the rabbit and nonhuman primate (rhesus) models

STUDY DESIGN: Cadaveric anatomic and in vivo survival animal studies were performed to develop a new arthrodesis technique for the lumbar spine. OBJECTIVES: To examine the feasibility, efficacy, and safety of a minimally invasive lumbar intertransverse process arthrodesis technique using an osteoinductive growth factor (rhBMP-2) delivered in a collagen sponge carrier. The technique was first developed using a rabbit model and modified for the nonhuman primate (rhesus monkey), a larger animal with the most similar bone biology to humans. SUMMARY OF BACKGROUND DATA: The morbidity of conventional posterolateral lumbar intertransverse process arthrodesis includes graft donor site morbidity; paraspinal muscle devascularization, denervation, and scarring and nonunion in up to 36% of patients. Minimally invasive anterior lumbar interbody arthrodesis techniques have been developed, but these often require a metal implant and carry risks to major vessels and development of retrograde ejaculation. A minimally invasive technique for posterolateral intertransverse process arthrodesis has not been described previously. METHODS: In Part 1, we examined seven New Zealand white rabbits and five rhesus monkeys at necropsy and during nonsurvival surgeries to determine the best access routes and to develop special instruments for video-assisted lateral intertransverse process arthrodesis. In Part 2, 38 New Zealand white rabbits underwent L4-L5 intertransverse process arthrodesis: the "OPEN" group (n = 16) underwent a standard open muscle-splitting approach using rhBMP-2 (bone morphogenetic protein) and collagen as a bone graft substitute; the "video-assisted control" group (n = 6) underwent video-assisted lateral intertransverse process arthrodesis with the collagen implant only (no growth factor); and the "video-assisted-BMP" group (n = 16) underwent video-assisted lateral intertransverse process arthrodesis with rhBMP-2 and collagen as the graft material. In Part 3, rhesus monkeys (n = 4) underwent video-assisted lateral intertransverse process arthrodesis using rhBMP-2 and collagen after laminectomy of L4-L5. RESULTS: In Part 1, we identified expedient, minimally invasive routes to the intertransverse process interval appropriate for each species: an intermuscular approach for the rabbit and a lateral, extramuscular approach for the rhesus monkey. In Part 2, all rabbits in the OPEN and video-assisted-BMP groups achieved solid intertransverse process lumbar fusions when assessed at 10 weeks. There were no neurologic impairments nor any difference between the two groups in the frequency of postoperative infection or other complications. None of the animals in the video-assisted control group showed evidence of fusion. In Part 3, exposure, decortication and grafting with rh-BMP-2 and collagen was accomplished successfully in all four monkeys through the video-assisted minimally invasive approach without complications. CONCLUSION: Video-assisted lateral intertransverse process arthrodesis is a feasible, effective, and safe method of lumbar spinal fusion in the rabbit and rhesus monkey. Use of this arthrodesis procedure will minimize the morbidity of paraspinal muscle denervation and devascularization seen with open intertransverse process fusion techniques, and the use of an osteoinductive growth factor will eliminate the problem of graft donor site morbidity and possibly increase the chances for successful fusion.     

Home visiting the newborn baby as a basis for developmental surveillance at child welfare centres

Home visiting is a part of the Swedish child health surveillance programme. In the present study, part of a longitudinal prospective project, the predictive power of observations at home visits to 338 newborn babies was evaluated. The regular home visit was made by the nurse at a Child Welfare Centre also using a check-list developed for this project. Her check-list assessments seemed valid in identifying families with stressful psychosocial conditions. When the general home situation was judged as "poor" or "dubious", the boys showed signs of a delayed mental development at 4-5 years of age. Assessments of "feeding problems" among boys were associated with behavioural problems at 4-5 years of age. The results underline the importance of an early home visit as a base for the developmental surveillance at Child Welfare Centres. However, the results of the home visit observations were not followed by any extra interventions at CWC. It seems the nurse should feel confident in her check-list judgement and initiate interventions where appropriate.     

Importance of localized skin infection in tick-borne encephalitis virus transmission

Arboviruses are transmitted to vertebrates by the "bite" of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfected Ixodes ricinus ticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels > or = 2.0 log10 ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission.     

Evidence for transient composition variations at GaAs/Ga1−xAlxAs heterostructure interfaces prepared by metal-organic chemical vapour deposition

The composition profiles of GaAs/Ga_1?xAl_xAs heterostructures prepared in two different Metal-Organic Chemical Vapour Deposition (MOCVD) reactors have been studied. Transmission electron microscopy (TEM). and Auger and secondary ion mass spectrometry (SIMS) sputter profiling results are in good agreement and interface widths below 20 å have been achieved. Significant new results on transient phenomena have been obtained. showing that large and sometimes very sharp excursions in composition can be associated with valve switching actions. Direct evidence is presented that gas pressure or flow transients can occur during valve operations. and it is suggested that such instabilities are the cause of the effects observed.