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.

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.

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.

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.

Parathyroid localization.

Twenty-nine consecutive patients with suspected primary hyperparathyroidism were examined preoperatively using ultrasound, sonographically guided fine needle aspiration, and aspirate immunostaining for PTH. In 25 patients, localization of enlarged parathyroid glands was successful. In 2 patients, the tumors were located retrosternally and, thus, could not be detected by ultrasound. One patient had a multinodular goiter which impeded localization. In 1 patient with renal osteodystrophy, 2 enlarged parathyroid glands in the neck were not visualized preoperatively. Cytology was not diagnostic, although some cytological features were suggestive of parathyroid cells. Immunostaining of the aspirated smears for PTH, however, correctly diagnosed all preoperatively localized lesions. Ultrasound should be the routine procedure of choice for preoperative localization of abnormal parathyroid glands in primary hyperparathyroidism. Fine needle aspiration and immunocytochemistry can supply confirmation, if necessary.

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.

Glutathione and GSH-dependent enzymes in the gastrointestinal mucosa of the rat.

The mucosal glutathione content of the gastrointestinal wall amounted to 50-60% of its concentration in the liver. GSH S-aryltransferase activity (CDNB) was very low in the glandular stomach, colon and rectum amounting to only 5% of liver enzyme activity. There was a marked postpyloric increase in GSH S-aryltransferase activity with an oral-aboral decline along the small intestine. GSH peroxidase was much lower in the mucosa of the small and large intestine as compared to the stomach or liver, whereas GSSG reductase was more than twice as high in the gastrointestinal mucosa as compared to the liver showing a gradual increase in activity from proximal to distal segments. The low GSH S-transferase activities found in the stomach, colon and rectum may account for the high and exclusive susceptibility of these segments to carcinogenesis and the deficient inducibility of these enzymes in the gastrointestinal wall may reflect an insufficient adaption towards higher exposure to toxic or even carcinogenic xenobiotics.

Aldrin epoxidase and dimethylhydrazine demethylase activities in tumorous and non-tumorous tissue of the human colon and rectum.

High activities of aldrin epoxidase and dimethylhydrazine demethylase were found in human colon and rectum mucosa, the first being as high as in human liver biopsy specimens. Comparisons between tumorous (adenocarcinomas) and non-tumorous tissues of the same individuals revealed loss of activities in tumorous specimens. The presence of epoxidizing enzymes and demethylation of the organ-specific colon carcinogen dimethylhydrazine (DMH) in the intestinal muscosa of tumor-bearing patients implicate them with chemical carcinogenesis also in humans.

Glutathione and GSH-dependent enzymes in the tumorous and nontumorous mucosa of the human colon and rectum.

A high content of total glutathione and high activities of both GSH S-aryltransferase (CDNB) and GSH peroxidase were found in different segments of the human intestinal mucosa comparable to findings in human gastric mucosa. Intraindividual comparisons of tumorous and nontumorous tissue specimens in patients with adenocarcinomas of the colon and rectum revealed no marked differences in their glutathione content and enzyme activities except in the sigma, where we found significantly lower GSH concentrations and higher GSH S-aryltransferase activities in the carcinomatous tissue. gamma-Glutamyl-transpeptidase activity, a marker of neoplastic cell growth in experimental hepatocarcinogenesis, did not differ between tumorous and nontumorous tissue areas. The presence and high activity of the GSH-dependent enzyme system in different segments of the human intestinal mucosa may reflect its role in the defense against toxic and putative carcinogenic xenobiotics entering the body via the gastrointestinal tract.

Susceptibility of Pasteurella haemolytica to the bactericidal effects of serum, nasal secretions and bronchoalveolar washings from cattle.

Several isolates of logarithmic-phase organisms of Pasteurella haemolytica were shown to be sensitive to an antibody and complement-mediated killing mechanism in adult bovine serum. Data suggested that the classical complement pathway was important in the induction of bactericidal activity of serum. Sera from calves after colostrum feedings (post-colostral sera) killed only 30% of the bacteria in spite of the presence of high levels of antibodies against P. haemolytica. Addition of post-colostral serum to heat-inactivated adult bovine serum decreased the bactericidal capacity of the latter. It was speculated that this inhibition may have been caused by the presence of blocking antibodies (IgA) found in the post-colostral serum. Undiluted nasal secretions collected from adult cattle were not bactericidal to P. haemolytica. The results also suggest that the bronchoalveolar washings (BAW) from vaccinated calves, in spite of having a high antibody titer, were less bactericidal to P. haemolytica than BAW from sham-vaccinated calves (71.12% vs. 83.12%). The bactericidal factor(s) present in BAW from sham-vaccinated calves was heat stable, not complement dependent, and was not related to lysozyme concentration.

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.

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.

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.

Studies on Pasteurella multocida. III. In vitro assay for cell-mediated immunity.

Peripheral blood lymphocytes (PBL) from turkeys immunized against fowl cholera with a bacterin or a live avirulent vaccine (strain CS-148) were cultured in vitro with various antigenic preparations from Pasteurella multocida (strain P-1059). The degree of lymphocyte stimulation (blastogenesis) was quantitated by measurement of the uptake of (3H) thymidine. Higher stimulation indices were obtained with immune lymphocytes rather than nonimmune lymphocytes. Stimulation was specific since PBL from turkeys immunized against P. multocida failed to react with Escherichia coli or Mycoplasma synoviae antigens. These differences were statistically significant as analyzed with the student's t-test. The lymphocyte transformation assay was emphasized as a convenient and useful in vitro indicator of cell-mediated immunity that should help define the role of cell-mediated immunity in P. multocida infections of turkeys.

Pasteurella multocida antigen-induced in vitro lymphocyte immunostimulation, using whole blood from cattle and turkeys.

A whole blood lymphocyte stimulation assay to study cell-mediated immune responses in bovine pasteurellosis was developed. Peripheral blood lymphocytes from cattle artificially immunised with three Asiatic haemorrhagic septicaemia strains of Pasteurella multocida exhibited higher stimulation indices when incubated with antigen preparations from homologous strains than with the heterologous shipping fever strain. Lymphocytes from cattle immunised with the shipping fever strain of P multocida exhibited a higher stimulation index when incubated with an antigen preparation from the homolgous strain than with antigen preparations from heterologous haemorrhagic septicaemia strains. These results suggest that immunogenic differences exist between the haemorrhagic septicaemia strains and the shipping fever strain of P multocida. An assay using turkey whole blood lymphocytes was also developed. The use of small amounts of whole blood, microtitre plates, either 125I iododeoxyuridine or 3H-thymidine as the labelling agent, and a multiple cell-culture harvester makes the method simple, rapid and suitable for the study of immune competence and cell-mediated immune responses in turkeys on a flock basis.

Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A.

A microculture system in conjunction with a semiautomatic multiple sample harvester (SAMSH) was used to study the in vitro properties of chicken peripheral lymphocytes. This new procedure enabled doing rapid multiple tests, using relatively few cells, and was highly reproducible. Data were presented to show many variables that are involved in studying the concanavalin A (Con A) response of chicken lymphocytes in a microculture system. Analysis indicated that the conditions for optimal Con A stimulation as measured by incorporation of 3H-TdR include: (a) use of 2 x 10(6) cells per culture in RPMI 1640 culture medium in the absence of any serum, (b) use of 0.4 mug of Con A per culture, (c) incubation at 37 degrees C for 72 hours, and (d) addition of 1 muCi of 3H-TdR to each culture 12 to 24 hours prior to termination. This technique could be used to monitor immunocompetence of the chicken.

A micromethod for evaluating turkey lymphocyte response to phytomitogens.

A microculture system used in conjunction with a semiautomatic sample harvester is described to determine the in vitro properties of turkey peripheral blood lymphocytes. By this new procedure, multiple tests were done rapidly, relatively few cells were used, and results were highly reproducible. Analysis indicated that the conditions for optimal concanavalin A (con A) stimulation, as measured by incorporation of 3H-thymidine (3H-TdR) included (a) 2 times 10(6) cells per culture in RPMI 1640 medium in the absence of any serum; (b) 0.4 mug of con A per culture, incubated in flat-bottom microtitration wells for 72 hours at 37 C; and (c) 1 muCi of 3H-TdR per culture added 12 to 24 hours before termination. Conditions for optimal stimulation with pokeweed mitogen were similar to those used for con A. The exceptions were that 1 times 10(6) cells per culture were incubated in round-bottom microtitration wells. The pokeweed mitogen gave the highest degree of stimulation when used at a concentration of 80 mug per culture.

Isolation of the turkey heterophil and measurement of its migratory functions under agarose.

A procedure for isolation of the turkey heterophil was developed. The procedure involves preliminary lysis of erythrocytes, using ammonium chloride and centrifugation of the leukocytes over a discontinuous gradient of 18% and 24% Ficoll. A minimum of 30% of the heterophils was consistently recovered from whole blood by application of this procedure. Spontaneous and chemotactic migratory responses of the isolated heterophils were assessed, using the migration under agarose technique. Supplementation of the agarose with 10% heat-inactivated turkey plasma and extension of the incubation period to 3 hours at 41 C in a 5% CO2-humidified atmosphere were required for optimal chemotaxis in response to zymosan-activated chicken serum. The heterophils did not respond chemotactically to the oligopeptide attractant, N-formyl-methionyl-leucyl-phenylalanine, and did not specifically bind a radiolabeled form of this ligand.

[Therapy of recurrent colorectal cancers]. / Zur Therapie der Rezidive colorectaler Carcinome.

Colorectal cancers treated for cure by operative means show tumour recurrences and metastases in about 20% of the patients. Of these colon or rectal cancers 75 and 90% respectively can be only operated for palliation. These patients have a better prognosis than those operated primarily for palliation. 25% of the cases especially with suture line recurrences can be operated upon radically enough and exhibit a total five year survival rate of 50%.

[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.