Dilution rate and pH effects on the conversion of oleic acid to trans C18:1 positional isomers in continuous culture.

In a previous in vitro study, mixed ruminal microorganisms converted oleic acid to a variety of trans monenes when grown in batch cultures under constant environmental conditions. To determine whether a similar conversion occurs under environmental conditions more typical of the rumen, conversion of 13C-labeled oleic acid to biohydrogenation intermediates was determined in ruminal microorganisms grown in continuous culture at two pH (5.5 and 6.5) and liquid dilution rates (0.05 and 0.10/h) arranged factorially. After each morning feeding of the dual-flow continuous cultures, 250 mg of oleic acid in 5 mL of ethanol were injected into each culture. On d 10, 250 mg of oleic-1-(13C) replaced the unlabelled oleic acid in ethanol. Trans fatty acids were isolated from culture samples by solid phase extraction, and 13C enrichment and identity of double bond position was determined by gas chromatography-mass spectroscopy. At pH 6.5 and 0.10/h dilution rate, 13C enrichment was detected in all trans-C18:1 isomers having double bond positions from C6 through C16 in the acyl chain. However, when pH or dilution rate in fermentors was lowered, no 13C enrichment was detected in any trans isomer with a double bond position beyond C10. Enrichment in stearic acid increased by reducing culture pH from 6.5 to 5.5, but decreased when dilution rate dropped from 0.10 to 0.05/h. The stearic acid carbons that originated from oleic acid biohydrogenation increased from 30 to 72% when pH dropped from 6.5 to 5.5. The 13C enrichment of trans-10 was reduced under low pH and dilution rate conditions. The results of this study confirm that ruminal microorganisms are capable of converting oleic acid to a wide variety of trans-C18:1 positional isomers when ruminal conditions are favorable (such as the pH 6.5 and 0.10/h dilution rate treatment). However, at low pH and dilution rate, the conversion of oleic acid to trans-C18:1 still occurs, but positional isomers produced are restricted to double bond positions from C6 to C10. Low pH conditions also increased the conversion of oleic acid to stearic acid.

Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses.

Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimer's disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimer's disease.

Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress.

We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (approximately 80%) and slowly back (approximately 20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein-tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimer's disease.

Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples.

Two direct methylation procedures often used for the analysis of total fatty acids in biological samples were evaluated for their application to samples containing fatty amides. Methylation of 5 mg of oleamide (cis-9-octadecenamide) in a one-step (methanolic HCl for 2 h at 70 degrees C) or a two-step (sodium methoxide for 10 min at 50 degrees C followed by methanolic HCl for 10 min at 80 degrees C) procedure gave 59 and 16% conversions of oleamide to oleic acid, respectively. Oleic acid recovery from oleamide was increased to 100% when the incubation in methanolic HCl was lengthened to 16 h and increased to 103% when the incubation in methoxide was modified to 24 h at 100 degrees C. However, conversion of oleamide to oleic acid in an animal feed sample was incomplete for the modified (24 h) two-step procedure but complete for the modified (16 h) one-step procedure. Unsaturated fatty amides in feed and digesta samples can be converted to fatty acid methyl esters by incubation in methanolic HCl if the time of exposure to the acid catalyst is extended from 2 to 16 h.

Neuronal growth cone collapse triggers lateral extensions along trailing axons.

Axonal outgrowth is generally thought to be controlled by direct interaction of the lead growth cone with guidance cues, and, in trailing axons, by fasciculation with pioneer fibers. Responses of axons and growth cones were examined as cultured retinal ganglion cell (RGC) axons encountered repellent cues. Either contact with cells expressing ephrins or mechanical probing increased the probability of lead growth cone retraction. Lateral extension of filopodia and lamellipodia hundreds of microns behind the lead growth cone was correlated with its collapse. Transmission electron microscopy showed that some of the lateral extensions originate from the pioneer axon, whereas others represent growth cones of defasciculating trailing axons.

Cellular localization of ephrin-A2, ephrin-A5, and other functional guidance cues underlies retinotopic development across species.

Avian retinotectal and rodent retinocollicular systems are general model systems used to examine developmental processes that underpin topographically organized neuronal circuits. The two systems rely on guidance components to establish their precise retinotopic maps, but many cellular events differ during their development. For example, compared with the chick, a generally less restricted outgrowth pattern is observed when retinae innervate their targets in rodents. Cellular or molecular distributions of guidance components may account for such differences in retinotopic development across species. Candidate repellent molecules, such as ephrin-A2 and ephrin-A5, have been cloned in both chick and rodents; however, it has not yet been shown in rodents that living cells express sufficient amounts of any repellent components to deter outgrowth. We used a coculture assay that gives cellular resolution of retinotarget interactions and demonstrate that living, caudal superior colliculus cells selectively prevent extension of axons from temporal regions of the retinae. Time-lapse video microscopy revealed the cellular localization of permissive and repulsive guidance components in rodents, which differed from that in chick. To analyze the potential molecular basis for these differences, we investigated the function and localization of ephrin-A2 and -A5. Cells transfected with ephrin-A2 and -A5 selectively repelled retinal axons. Ephrin-A2 and -A5 RNA expression patterns differed across cell populations and between species, suggesting molecular mechanisms and key cellular interactions that may underlie fundamental differences in the development of retinotectal and retinocollicular maps.

Plasma fatty acids in sheep fed hydroxyethylsoyamide, a fatty acyl amide that resists biohydrogenation.

Hydroxyethylsoyamide (HESA) was made from soybean oil (SBO) and ethanolamine to determine the effectiveness of this fatty acyl amide to escape ruminal biohydrogenation and increase unsaturated fatty acids in plasma of sheep. The disappearance rate of 18:2n-6 from in vitro cultures was reduced 61% when the substrates contained added HESA compared to added linoleic acid. The decline in acetate to propionate ratio in the cultures also was less for HESA than for linoleic acid, which indicates lower inhibition of fermentation by HESA. Four sheep were housed in metabolism stalls for the collection of feces and urine and fed four diets in a Latin square design with 17-d periods. The diets contained no added fat (control) or were supplemented with 2.5% SBO, 5% butylsoyamide (BuSA), or 5% HESA. Plasma 18:2n-6 was not changed by feeding SBO. However, compared to the control diet, 18:2n-6 increased 19 and 35% in plasma fatty acids, and 74 and 113% in plasma triglycerides when sheep were fed BuSA and HESA, respectively. Digestibility of HESA was greater than for BuSA (99.5 vs. 48.4%, respectively). No amide was detected in plasma of sheep fed either BuSA or HESA. This study further supports that unsaturated oils can be converted to fatty acyl amides as a way to avoid ruminal biohydrogenation and elevate unsaturated fatty acids in body tissues of ruminants. Also, digestibility of the amide and plasma unsaturated fatty acids was enhanced more when the amide was synthesized from ethanolamine than when synthesized from butylamine.

Localized amyloid in the menisci of the knee joint.

This is, to the best of our knowledge, the first report on amyloid deposits in menisci. Fragments of menisci gained by arthroscopy from 316 patients between 20 and 80 years of age were examined. Amyloid was found in 70% of the cases from male, as well as female patients. The amyloid amount found was always very small, but the deposits seemed to increase with age. Patients more than 50 years of age all had menisceal amyloid. Two types of deposits were observed: a)stroma-deposits in the deep central portions of the menisci (tiny dots of intensely stained amyloid and/or ill defined patches of low staining intensity) and b) surface associated deposits: band-like amyloid imbibition of the collagenous stroma immediately beneath the surface of the menisci but not deeper than 0.2 mm. In all cases, amyloid was resistant when pretreated by KMn04 and immunohistologically antisera against amyloid types AA, AB and AF were negative. 3/25 cases showed a reaction with an amyloid-lambda-antibody. We assume, that amyloid in menisci is a further type of localized senile amyloidosis.

Cellular localization of guidance cues in the establishment of retinotectal topography.

Topographic projections of the nervous system are essential to numerous brain functions. They arise during development as a result of encounters between projecting growth cones and particular target cells. Cellular localization of guidance cues can indicate the sequential processes involved in establishment of such topography. The map formed by retinal ganglion cells on their target nuclei has served widely as a model system to investigate mechanisms underlying the highly precise and stereotypic connectivity of the nervous system. To investigate cellular localization of guidance cues in the developing retinotectal system, a three-compartment chamber was created to delimit areas where cultured embryonic chick retinal ganglion axons and tectal cells encounter one another and guidance behavior could be readily assessed. Whereas explants from nasal retinae extended fibers across their natural target population, fibers from temporal regions of retinae failed to invade areas of growing posterior tectal cells. This preservation of relevant guidance information on living cell populations enabled an evaluation of retinal ganglion cell growth cone behavior after encounter with individual tectal cells. Posterior tectal neurons appeared selectively repulsive for temporal retinal ganglion cell growth cones, causing growth cone collapse and retraction. On the contrary, neuroepithelial cells from all regions of the tectum attenuated retinal ganglion axon extension, without inducing sudden retraction. Nasal growth cones traversed or tracked more often along neuroepithelial cells from their natural target area, potentially indicating a second set of guidance cues possibly localized to posterior glia. Together, these differential interactions suggest that development of retinotectal topography critically depends on cell-specific cues, which are distributed selectively on particular populations of target cells.

Replacing cis octadecenoic acid with trans isomers in media containing rat adipocytes stimulates lipolysis and inhibits glucose utilization.

On the basis of earlier reports of reduced growth rate and fat accumulation in animals fed trans 18:1, a study was conducted to determine whether the effects of trans 18:1 on lipolysis and glucose utilization by adipocytes differed from effects of the cis isomer. Two experiments compared three fatty acid isomers (oleic, elaidic and vaccenic acids) at several concentrations and at several fatty acid to albumin ratios in cell media. Adipocytes were isolated from adipose tissue of rats by collagenase digestion and incubated for 2 h in media containing added fatty acids. Compared with oleic acid, both trans isomers reduced (P < 0.01) the amount of glucose converted to cell lipid in both experiments. Glucose oxidation to carbon dioxide also was lower for both trans fatty acids in Experiments 1 (P < 0.05) and 2 (P < 0.06). Lipolytic rates were increased (P < 0.01) in both experiments by replacing oleic acid with either of the trans isomers. Trans isomers of octadecenoic acid had catabolic effects on adipocyte metabolism that occurred regardless of the position of the double bond, the fatty acid concentration in media or the fatty acid to albumin ratio. These catabolic effects explain previous observations of reduced growth rate and fat accumulation when oleic acid in animal diets is replaced with a trans isomer.

Dietary soybean oil changes lipolytic rate and composition of fatty acids in plasma membranes of ovine adipocytes.

A study was conducted to determine which fatty acids in plasma membranes of adipose tissue from ruminants are changed when the diet is supplemented with unsaturated fatty acids and to determine the effect of the fat supplement on adipocyte metabolism. Ten sheep were randomly assigned to two isonitrogenous diets containing either no added fat (control) or 5 g soybean oil/100 g diet. Perirenal fat was removed at slaughter, adipocytes isolated by collagenase digestion, and plasma membranes prepared by centrifugation on a Percoll gradient. Feeding soybean oil to the sheep increased (P < 0.05) linoleic acid [18: 2(n-6)] concentration in subcutaneous fat and isolated adipocytes, suggesting partial escape of dietary unsaturated fatty acids from ruminal biohydrogenation. Soybean oil consumption also decreased (P < 0.05) concentrations of myristic acid, arachidonic acid [20: 4(n-6)] and anteiso 17:0 in plasma membranes, but increased (P < 0.05) trans 18:1. Lipogenesis was not affected by diet, but lipolysis tended to be greater (P = 0.07) in sheep fed the soybean oil-containing diet than in those fed the control diet. In ruminants, fatty acids of ruminal origin, namely trans intermediates of biohydrogenation or branched-chain fatty acids of microbial lipid, may account for as much change in the composition of plasma membranes and in cellular metabolism as do the small quantities of unsaturated fatty acids in the diet that escape biohydrogenation.

Effects of the detergent Tween 80 on Thermomonospora curvata.

Tween 80 (0.1%, v/v) added to Thermomonospora curvata growing in minimal medium caused a transient lowering of the dry cell mass, decreased the optimal growth temperature of the thermophile from 62 to 54°C, and increased extracellular esterase activity. Cells grown in the presence of Tween 80 had decreased concentrations of branched chain fatty acids and increased concentrations of oleic acid. The detergent removed surface protuberances from mycelia and increased the liberation of enzymes active against crystalline cellulose, but did not stimulate liberation of enzymes active against carboxymethylcellulose, starch or pectin.

Appearance of target-specific guidance information for regenerating axons after CNS lesions.

During development of the vertebrate visual system, an orderly projection of ganglion cells from the retina onto the superior colliculus (SC) is established. Mechanisms that might govern this process include the coordinated action of guidance and corresponding receptor molecules that are specifically distributed on the axons and their targets. In birds and mammals, information for axonal guidance and targeting appears to be confined to the time when the retinocollicular projection is being formed. Here we show that putative guidance activities for temporal and nasal retinal axons, which are not detectable in the normal adult SC, appear after optic nerve transection in adult rats. Both embryonic and adult retinal axons are able to respond to these guiding cues, although the guidance activities detectable in the deafferented adult rat SC might be different from those found during development. These findings imply that it might be possible to reestablish an ordered projection after lesions in the adult mammalian visual system.

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

[Traumatic false aneurysm of the subclavian artery]. / Das traumatische Aneurysma spurium der Arteria subclavia.

Injuries of the subclavian artery are rare; still rarer are aneurysms of the subclavian artery due to blunt or penetrating trauma. Diagnosis can be performed by careful clinical investigation. To identify precisely the nature and site of the injury, angiography and CT are necessary. Rarely associated injuries, such as arteriovenous fistulas, can be recognized by means of these diagnostic tools. This will be of value for planning the surgical approach, which varies with the anatomical site of the injury and the kind of accompanying damage. Subclavian aneurysms should be treated surgically, because embolic or thrombotic complications may threaten the extremity and the brain.

Chromosome jumping from flanking markers defines the minimal region for alf/hsdr-1 within the albino-deletion complex.

The locus alf/hsdr-1, defined by the albino-deletion complex on mouse chromosome 7, is essential for neonatal survival. Animals homozygous for a subset of the deletions die shortly after birth due to impaired gene expression in liver parenchymal cells and kidney proximal tubular cells. Here, we describe a detailed analysis of the region containing alf/hsdr-1 by means of chromosome jumping from flanking markers. Three chromosome jumping libraries based on the restriction enzymes XmaI and SalI were constructed. Isolation of eight jumping clones distributed over 450 kb allowed more than 240 kb to be cloned in genomic lambda and cosmid libraries. Five of the probes map within the minimal genetic interval for alf/hsdr-1, which is defined by the proximal borders of the deletions c10R75M and c11DSD. The breakpoints of these deletions were precisely mapped, which allowed alf/hsdr-1 to be localized to a 310-kb interval.

Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice.

Mice homozygous for albino deletions encompassing the locus alf/hsdr-1 die shortly after birth. Lethality is thought to be the consequence of hypoglycemia, which results from the failure to activate hormone-dependent genes in liver and kidney encoding enzymes important for gluconeogenesis. Within the region in which alf/hsdr-1 has been defined by physical mapping, we identified the gene encoding fumarylacetoacetate hydrolase (FAH), an enzyme of tyrosine metabolism. Lack of FAH activity should lead to accumulation of toxic tyrosine metabolites. In man, genetically determined FAH deficiency is the primary defect in tyrosinemia type I, a fatal liver disease of infants. Northern blot and in situ hybridization analysis of mouse tissues showed that the cell types that normally express FAH correspond to those that exhibit a phenotype in alf/hsdr-1 deletion mice. Moreover, we could mimic aspects of the alf/hsdr-1 deletion phenotype in vitro by treating primary hepatocyte cultures with an intermediate of tyrosine metabolism. These findings strongly suggest that alf/hsdr-1 encodes FAH and that absence of FAH is responsible for neonatal lethality in albino deletion mice. Mechanisms by which this metabolic defect might bring about alterations in gene expression characteristic of the alf/hsdr-1 deletion phenotype are discussed.

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.