Parvalbumin-containing interneurons of the human cerebral cortex express nicotinic acetylcholine receptor proteins.

Cholinergic fibers from the basal forebrain are known to contact cholinoceptive cortical pyramidal neurons. Recent electrophysiological studies have revealed that nicotinic acetylcholine receptors are also present in human cerebrocortical interneurons. A direct visualization of nicotinic receptor subunits in cortical interneurons has, however, not yet been performed. We have applied double-immunofluorescence using antibodies against parvalbumin --a marker for the Chandelier and basket cell subpopulation of interneurons--and to the alpha4 and alpha7 subunit proteins of the nicotinic acetylcholine receptor. The vast majority of the parvalbuminergic interneurons was immunoreactive for the alpha4 and the alpha7 nicotinic acetylcholine receptor. Provided these receptors would be functional--as suggested by recent electrophysiological findings--the connectivity pattern of cholinergic afferents appears much more complex than thought before. Not only direct cholinergic impact on cortical projection neurons but also the indirect modulation of these by cholinergic corticopetal fibers contacting intrinsic cortical cells would be possible.

All three PACSIN isoforms bind to endocytic proteins and inhibit endocytosis.

The PACSINs are a family of cytoplasmic phosphoproteins that play a role in vesicle formation and transport. We report the cloning and cDNA sequencing of PACSIN 3 and the analysis of all three PACSIN isoforms with regard to tissue distribution, ligand binding properties and influence on endocytosis. PACSIN 3 differs from the other family members in having a short proline-rich region and lacking asparagine-proline-phenylalanine motifs. In contrast to the neurospecific PACSIN 1 and the ubiquitously expressed PACSIN 2, PACSIN 3 is mainly detected in lung and muscle tissues. All isoforms potentially oligomerize and bind to dynamin, synaptojanin 1 and N-WASP via their Src homology 3 domains. The PACSIN proteins colocalize with dynamin, but not with clathrin, implying a specific role with a distinct subpopulation of dynamin at defined cellular sites. Transferrin endocytosis is blocked in a dose-dependent manner in cells overexpressing the PACSIN variants, but the inhibitory effect can be abolished by mutating specific amino acid residues in the Src homology 3 domains. These characteristics of the PACSIN protein family suggest a general function in recruitment of the interacting proteins to sites of endocytosis.

Expression of nicotinic acetylcholine receptors in Alzheimer's disease: postmortem investigations and experimental approaches.

Nicotinic ligand binding studies have shown rather early that the cholinoceptive system is affected in Alzheimer's disease (AD). Today, molecular histochemistry enables one to study the nicotinic acetylcholine receptor (nAChR) subunit expression on the cellular level in human autopsy brains, in animal models and in in vitro approaches, thus deciphering the distribution of nAChRs and their role as potential therapeutic targets. The studies on the nAChR expression in the frontal and temporal cortex of AD patients and age-matched controls could demonstrate that both, the numbers of alpha4- and alpha7-immunoreactive neurons and the quantitative amount, in particular of the alpha4 protein, were markedly decreased in AD. Because the number of the corresponding mRNA expressing neurons was unchanged these findings point to a translational/posttranslational rather than a transcriptional event as an underlying cause. This assumption is supported by direct mutation screening of the CHRNA4 gene which showed no functionally important mutations. To get more insight into the underlying mechanisms, two model systems organotypic culture and primary hippocampal culture - have been established, both allowing to mimic nAChR expression in vitro. In ongoing studies the possible impact of beta-amyloid (Abeta) on nAChR expression is tested. Preliminary results obtained from primary cultures point to an impaired nAChR expression following Abeta exposure.

Classical Alzheimer features and cholinergic dysfunction: towards a unifying hypothesis?

OBJECTIVE: Our autopsy studies show possible links between classical Alzheimer pathology and decreased expression of nicotinic acetylcholine receptors. For further elucidation we are now using in vitro models. We report preliminary evidence for the impact of beta-amyloid on nicotinic receptor expression in hippocampal dissociation culture. METHODS: Cultures (E18 rats) were grown in a serum-free medium and incubated at 8 days in vitro for 3 days with 1 microM Abeta1-42. Expression of alpha4, alpha7, and beta2 nicotinic receptor subunit protein was assessed immunohistochemically and rated semiquantitatively. RESULTS: Abeta1-42 incubation resulted in a massive reduction of alpha4 protein-expressing neurons, this effect was less pronounced for the alpha7 and beta2 subunit protein. CONCLUSION: These findings provide first evidence for a direct impact of classical Alzheimer pathology features on nicotinic receptor expression in vitro. Our model will be useful for testing the potential of drugs to stop or reverse these effects.

[Prevention of postoperative ischemic colitis in aorto-iliac vascular reconstruction]. / Prophylaxe der postoperativen ischämischen Colitis bei aorto-iliacaler Gefässrekonstruktion.

In consecutive series with 1740 aorto-iliac reconstructions (1970-1989) 32 patients (1.8%) with postoperative ischemic colitis have been registered. In a retrospective follow-up (1970-1983, group I, n = 1121) the incidence of this postoperative complication (ischemic colitis grade C) reached to 0.7%. In a further prospective follow-up (group II, 1984-1989, n = 619) the operative procedure was influenced by: 1) intraoperative stump pressure in the inferior mesenteric artery, 2) by the penile brachial index (PBI), 3) pre- and postoperative rectoscopy, and 4) measurement of perfusion in branches in the inferior mesenteric artery. The frequency of ischemic colitis could not be reduced. Grade C colitis reached to 1.3%. Rectosigmoidoscopy as a routine method before and after operation caused an remarkable decrease of the time interval between operation and diagnosis of ischemic colitis in group II (from 11 days to 24/48 h). As a result the mortality rate of transmural colon necrosis (grade C) could be reduced from 50% (group I) to 13.6% (group II). The IMA stump pressure alone (limit of 40 mm Hg) doesn't help to decide if reimplantation of the IMA necessary. The stump pressure with Doppler ultrasound flow registration in the branches of the IMA gains a greater sensitivity by a "two vessel clamping" (infrarenal aorta and digital compression of the SMA) (sensitivity 100%). During the last 230 surgical interventions the incidence of ischemic colitis ranged 0.9%.

Porcine factor VIII and plasmapheresis in the management of hemophiliac patients with inhibitors.

Management of hemophiliac patients with inhibitors can be a difficult problem. We report here a patient with severe hemophilia A, a high titer inhibitor, and an abdominal abscess requiring surgery. Despite significant cross reactivity of the inhibitor with porcine FVIII, he was successfully managed with plasmapheresis and porcine FVIII infusions. Other approaches to the management of such patients are briefly reviewed.

Hydrolysis of endogenous phospholipids by rat brain microsomes.

Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl-3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate-labeled microsomes. In oleate-labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin-stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso-compounds. Mellitin and oleate together reduced the radioactivity found in lyso-PC and increased that in lyso-PE.

On the phospholipid metabolism of glial cell primary cultures. II. Metabolism of 1-alkyl-glycerophosphoethanolamine during time course.

Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-3H] alkyl-sn-glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1-20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmalogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl-compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.

On the phospholipid metabolism of glial cell primary cultures. III. Utilization of 1-alkenyl-glycerophosphoethanolamine (lysoplasmalogen).

Primary cultures prepared from newborn rat brain, consisted after 16 or 17 days mainly of astrocytes and of oligodendrocytes. 1-Alkenyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) was used as substrate for studies on the metabolism of ethanolamine-glycerophospholipids. After 3 hr incubation two main products were observed: a) 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (= ethanolamine plasmalogen) and b) 1-alkenyl-2-acyl-sn-glycero-3-phosphocholine (= choline plasmalogen). The acylation rate reached saturation at about 10 nmol substrate/mg cell protein with a Vmax of 30 nmol x mg cell protein-1 x 3 hr-1. This acylated compound amounted to almost 60% of all radioactivity internalized, whereas the second product, choline plasmalogen, came to 20%. Unchanged substrate was found within the cells only in small amounts, even at maximum substrate internalization. These results were discussed in comparison with those obtained with 1-alkyl-sn-glycero-3-phosphoethanolamine under the same conditions (25).

Phospholipid metabolism of glial cell primary cultures, IV. Metabolism of 1-alkenyl-sn-glycero-3-phosphoethanolamine between 1 and 20 hours incubation.

Primary cell cultures prepared from newborn rat brain were incubated on the 16th or 17th day with the substrate 1-([1-3H]-1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) for 1-20 h. The internalization of the substrate into the cells depended on the incubation time as well as on the amount of substrate. At any given time the acylation reaction to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine (plasmalogen) was the most important event amounting to nearly 50-60% of the total radioactivity incorporated. Unchanged substrate was found in only small amounts within the cells. During incubation, the formation of 2-acyl-1-alkenyl-sn-glycero-3-phosphocholine (choline plasmalogen) increased, reaching saturation after 6 h with nearly 40% of the total radioactivity within the cells. These results were compared with those previously obtained with the substrate 1-([1-3H]alkyl)-sn-glycero-3-phosphoethanolamine under the same conditions. The acylation of this substrate as well as its conversion to the choline-containing analogue had been observed. Furthermore plasmalogen formation was also determined as a slow enzyme reaction. Both series of experiments showed a high acylation rate of 1-alkenylglycerophosphoethanolamine and a slow desaturation rate of the 1-alkyl compound. Thus, the following pathway of plasmalogen formation is proposed: 1-alkyl-sn-glycero-3-phosphoethanolamine leads to 1-alkenyl-sn-glycero-3-phosphoethanolamine leads to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine.

On the phospholipid metabolism of glial cell primary cultures: cell characterization and their utilization of 1-alkyl-glycerophosphoethanolamine.

Primary cultures prepared from newborn rat brain were grown for 16 or 17 days in culture. Addition of brain extract from newborn rats to the medium stimulated the maturation of astrocytes and the development of oligodendrocytes. Both cell types were characterized by morphology and by immunohistochemistry. The phospholipid composition of these cells was estimated. Incubations were performed with 1-[3H]alkyl-sn-glycerophosphoethanolamine in varying concentrations for 3 h. About one-third of the substrate supplied was internalized by the cells. Several enzymic reactions were observed. The acylating enzyme system was the most active one--a Km was determined with 5 nmol intracellular 1-alkyl-sn-glycerophosphoethanolamine/mg cell protein. Plasmalogen formation was rather low. 1-Alkyl-sn-glycerol, a hydrolysis product, was found in small amounts. Some radioactivity was also incorporated into the phosphatidylcholine fraction.

Hairy cell leukemia and sarcoid.

A 45-year-old female simultaneously developed both hairy cell leukemia and acute sarcoid. A defect in suppressor T lymphocytes which allowed an unrestrained B-cell proliferation manifested as both hairy cell leukemia and sarcoid is proposed.