Onset of Kleine-Levin Syndrome in association with isotretinoin treatment.

UNLABELLED: The synthetic retinoid isotretinoin is an effective treatment option for severe forms of acne vulgaris. However, several reports indicate that some patients experience altered central nervous system functions in association with treatment. We present here the first description of the onset of Kleine-Levin Syndrome (KLS), a rare disorder characterised by periodic hypersomnia and cognitive and behavioural symptoms, in close temporal relation to the start of isotretinoin treatment. We also discuss the biological potential of retinoids to affect sleep. CONCLUSIONS: In light of a documented potential of retinoids to modulate sleep-wake regulation, the present case suggests that isotretinoin may rarely trigger the onset of KLS.

Binding of the periodontitis associated bacterium Porphyromonas gingivalis to glycoproteins from human epithelial cells.

INTRODUCTION: In the present study we examined the ability of the periodontal pathogen Porphyromonas gingivalis to adhere to glycoconjugates on intact cells and to protein preparations of epithelial cells (KB cells). METHODS: The KB cell protein preparation was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by Western blotting. The membranes were used in overlay assays with labeled P. gingivalis. Flow cytometry was used to analyze attachment of bacteria to intact KB cells. RESULTS: Glycoconjugate expression on the KB cells and in the protein preparation was confirmed. Binding was detected to several bands on the Western blots. Flow cytometry showed a distinct increase in fluorescence for strain FDC 381. Preincubation of the bacteria with mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine inhibited the binding to KB cells by approximately 30% whereas preincubation with N-acetylneuraminic acid reduced the binding by 60%. CONCLUSION: These results indicate that carbohydrate structures are involved in the binding process of P. gingivalis to oral epithelial cells and that neuraminic acid plays a significant role in the adhesion process.

Transplantation of preconditioned intestinal grafts is associated with lower inflammatory activation and remote organ injury in rats.

Reperfused grafts--particularly the intestine--release free radicals and cytokines into the systemic circulation. The type of discharge, which is greatly dependent on the local injury, may also induce inflammatory activation in distant organs and leading to multiple system and organ failure. It has been suggested that intestinal grafts from tacrolimus (TRL)-pretreated donors show improved morphology and microcirculation. We studied whether transplantation of intestines from TRL-pretreated donors influenced inflammatory response and remote organ injury posttransplantation. Donor Sprague Dawley rats received TRL or saline (controls) intravenously at 6 hours prior to graft harvest. The intestinal grafts were preserved in saline for 3 hours before transplantation. At 6 and 12 hours postreperfusion hepatic and renal cortical microcirculation were assessed using laser-Doppler flowmetry (n = 8-12 per group). Blood pressure was measured; liver, kidney, and serum samples were obtained. We analyzed hepatic and renal ICAM-1 expression and caspase-3-like activity as well as plasma content of tumor necrosis factor-alpha and interleukin-6. Pretreated graft recipients had higher mean arterial pressure (82 +/- 10 vs 51 +/- 17 mm Hg, P < .05) and renal perfusion at 6 hours whereas liver perfusion was similar at both 6 and 12 hours. Liver and renal functions were also superior among recipients of pretreated grafts. Both caspase-3-like activity and ICAM-1 expression in liver and kidney were lower in pretreated graft recipients. Plasma IL-6 levels were lower in animals receiving pretreated grafts. Transplantation of intestines from TRL-pretreated donors was followed by a lower systemic inflammatory response, improved organ function and decreased remote injury early posttransplantation compared with animals receiving grafts from untreated donors.

Oral feeding with pig peripheral lymphocytes decreases the xenogeneic delayed type hypersensitivity reaction in galactosyltransferase knockout mice.

PURPOSE: Oral tolerance induction has shown promising results in experimental allotransplantation models but is not well investigated in xenotransplantation. We investigated the possibility to induce tolerance against pig peripheral lymphocytes (pPBL) in galactosyltransferase knockout mice (gal -/-), which produce antibodies against Galalpha1-3Gal. MATERIAL AND METHODS: Female (gal -/-) mice 6 to 8 weeks old weighing 35 to 40 g (n = 10) were fed orally every third day five times with 2 x 10(7) isolated, viable pPBL, or with phosphate-buffered saline (PBS) only (n = 7). They were then immunized subcutaneously on day 0 with a subcellular lysate from 4 x 10(7) isolated, viable pPBL. On day 13, 25 microL of a subcellular lysate corresponding to 1 x 10(7) isolated, viable pPBL was injected in the right dorsal foot pad, and the delayed type hypersensitivity (DTH) reaction was calculated after 24 hours by subtracting the swelling response from 25 microL PBS in the left footpad. Anti-Galalpha1-3Gal immunoglobulin IgG and IgM antibody titers were measured in the serum before oral feeding and at day 14. RESULTS: The DTH reaction of the pPBL fed mice was 0.07 +/- 0.05 mm vs 0.57 +/- 0.23 mm for the controls (P < .001). No significant differences in anti Gal alpha1-3 Gal IgG and IgM antibody titers were seen. CONCLUSIONS: This study demonstrates for the first time that oral delivery of pPBL can counteract the indirect T-cell reaction against xenogeneic subcellular antigens from pPBL. These observations warrant further investigation in immunologically modified mice and perhaps in primate models of xenotransplantation.

Degradation of GFP-labelled POM121, a non-invasive sensor of nuclear apoptosis, precedes clustering of nuclear pores and externalisation of phosphatidylserine.

The nuclear pore membrane protein POM121 is specifically degraded during apoptosis by a caspase-3-dependent process enabling early detection of apoptosis in living cells expressing POM121-GFP. Here we further investigated temporal aspects of apoptotic degradation of POM121-GFP. We demonstrate that decreased POM121-GFP fluorescence precedes annexin V-labelling of apoptotic cells. This indicates that degradation of the nuclear pore complex starts prior to redistribution of plasma membrane phosphatidylserine, which serves as a signal for phagocytotic elimination of apoptotic cells. Furthermore, a caspase-resistant GFP-labelled mutant of POM121 resisted degradation even in late apoptosis and was detected in clustered nuclear pores. Thus, it can be concluded that loss of POM121-GFP is a specific sensor of the activation of caspase-3-dependent proteolysis at the nuclear pores.

Role of maxilla 2 and its setae during feeding in the shrimp Palaemon adspersus (Crustacea: Decapoda).

The movements of the basis of maxilla 2 in Palaemon adspersus were examined using macro-video recordings, and the morphology of its setae was examined using both scanning and transmission electron microscopy. The basis of maxilla 2 performs stereotypical movements in the latero-medial plane and gently touches the food with a frequency of 3-5 Hz. The medial rim of the basis of maxilla 2 carries three types of seta. Type 1 is serrate, type 2 and 3 are serrulate, and type 2 has a prominent terminal pore. Type 2 is innervated by 18-25 sensory cells whose cilia protrude through the terminal pore and are in direct contact with the external environment. The structure of type 2 setae indicates that they are mainly gustatory, although still bimodal due to their innervation by presumed chemosensory and mechanosensory neurons. Distally, the three types of setae have a complex arrangement of the cuticle involving water-filled canals, which may serve to improve flexibility. Type 1 and 3 setae have fewer sensory cells (4-9) but probably also have a bimodal sensory function. The function of type 1 setae is probably to protect type 2 setae, while type 3 setae might serve to groom the ventral side of the basis of maxilla 1.

Sequential degradation of proteins from the nuclear envelope during apoptosis.

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

A chiral sex pheromone system in the pea midge, Contarinia pisi.

The sex pheromone of the pea midge consists of 2-acetoxytridecane, (2S,11S)-diacetoxytridecane and (2S,12S)-diacetoxytridecane. The responses of male pea midges to the corresponding stereoisomers of (2S,11S)-diacetoxytridecane and (2S,12S)-diacetoxytridecane were tested in field trapping experiments and by electroantennographic recordings. When added at 20% of the pheromone component to the sex pheromone blend, the (2S,11R)- and (2R,11S)-stereoisomers of (2S,11S)-diacetoxytridecane, were shown to have a strong inhibitory effect on male attraction in the field. At the same dose, (2R,11R)-diacetoxytridecane, (2R,12R)-diacetoxytridecane, and meso-2,12-diacetoxytridecane, did not have a significant effect on male behavior. It was also shown that substitution of either (2S,11S)-diacetoxytridecane or (2S,12S)-diacetoxytridecane with the related stereoisomers reduced trap catches to the level of blank traps. The electroantennographic recordings showed similar dose-response curves for the pheromone components and the stereoisomers shown to have an inhibitory effect. It seems likely that male antennae have receptors for bothpheromone components and for inhibitory stereoisomers. Scanning electron microscopy and transmission electron microscopy of the antennae revealed three types of sensilla involved in chemoreception: sensilla circumfila, sensilla trichodea, and sensilla coeloconica. The sensilla circumfila and trichodea are both innervated by two sensory cells, whereas the sensilla coeloconica are innervated by four to five cells.

Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: implications for annulate lamella formation.

Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. Söderqvist et al., 1997, Eur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP(3)) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP(3) had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP(3) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP(3) into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0. 1 to 2 microm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.

Phagocytic and intestinal endothelial and epithelial barrier function during the early stage of small intestinal ischemia and reperfusion injury.

The effects of intestinal ischemia and reperfusion (I/R) on small intestinal mucosal endothelial and epithelial barrier integrity and phagocytic function were assessed in rats subjected to 20- or 40-min mesenteric ischemia and a 3-h reperfusion. The results showed that human serum albumin (125I-HSA) flux through the endothelial layer to the interstitial space increased as did 125I-HSA clearance from blood to the gut lumen and 131I-HSA flux from the gut lumen to the interstitial space in rats with I/R. E. coli adhering to microvilli, invading and passing into the microvessels, were noted on the small intestinal mucosa in animals subjected to 40-min ischemia and a 3-h reperfusion. Phagocytic function increased, especially in the small intestinal wall, lungs, liver, and spleen in the groups with I/R, correlating with the length of ischemia. The results imply that both endothelial and epithelial barrier integrity is impaired in the early phase after I/R and that the epithelial barrier more effectively restricts macromolecular leakage compared with the endothelial barrier. I/R impairs the intestinal barrier not only by causing tissue hypoxia but also by activating the phagocytic system and aggravating barrier damage, which finally may result in bacterial translocation and remote organ dysfunction.

Morphology of antennal sensilla auricillica and their detection of plant volatiles in the Herald moth, Scoliopteryx libatrix L. (Lepidoptera: Noctuidae).

In the Herald moth Scoliopteryx libatrix there are single superficial auricillic sensilla, as well as groups of s. auricillica located in cavities on the antennae. Two sensory neurones, with different dendrite diameters innervate each of these sensilla. The diameter of the smaller dendritic segment is roughly half that of the larger one. The larger dendritic outer segment branches profusely in the lumen of the sensillum, whereas the smaller dendrite has few branches. Electrophysiological recordings from s. auricillica located in the medial part of the cavity revealed a receptor neurone responding to Delta-3-carene. In addition to these neurones, recordings made deeper and more laterally into the cavity showed neurones that responded to (+/-)-linalool, alpha-pinene and green leaf volatiles.

Liquid chromatography "on-flow" 1H nuclear magnetic resonance on native glycosphingolipid mixtures together with gas chromatography/mass spectrometry on the released oligosaccharides for screening and characterisation of carbohydrate-based antigens from pig lungs.

Glycosphingolipids were prepared from pig lung and pooled into two fractions with (i) < or = 3 sugar residues, and (ii) > or = 3 sugar residues. Oligosaccharides were prepared and used for gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. The glycolipid fractions i and ii were further characterised and purified using a novel method based on high performance liquid chromatography "on-flow" proton nuclear magnetic resonance. The LC "on-flow" NMR technique showed good chromatographic separation and gave NMR spectral information which could be used as guidance for pooling of the separated mixture glycolipids. Conventional 1H NMR, thin layer immunostaining, gas chromatography, gas chromatography/mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry were used to characterise the glycolipids and to validate LC-NMR spectral data.

Arthropod sensilla: morphology and phylogenetic considerations.

The structures of different types of arthropod sensilla are compared and theories regarding the evolution of these sensory organs are presented. Arthropod sensilla are built according to a common plan, and are probably homologous to scolopidia. Certain similarities in the structure of sensilla in different arthropod groups can be the result of adaptations to specific environments. The structure of sensilla in insect groups, which are regarded to be ancestral, do not appear to be less sophisticated than in groups considered to be more advanced. The different types of pore systems, as well as the structural differentiations of insect olfactory sensillar types remain unexplained. Olfactory sensilla display a large degree of similarity among terrestrial arthropods, whereas crustacean sensilla diverge in structure. In holometabolous insects larval sensilla appear to be structurally quite advanced, and more complex than in the adult. During the ontogeny of both sensilla and scolopidia, these are differentiated in an epithelial layer, resulting in the formation of both sensory and enveloping cells. The developmental patterns of sensilla in the studied insect groups are similar. During the development of sensilla apoptotic process are usually active.

Intracellular trafficking of emerin, the Emery-Dreifuss muscular dystrophy protein.

Emerin is an integral protein of the inner nuclear membrane that is mutated or not expressed in patients with Emery-Dreifuss muscular dystrophy. Confocal immunofluorescence microscopy studies of the intracellular targeting of truncated forms of emerin, some of which are found in patients with Emery-Dreifuss muscular dystrophy, show that the nucleoplasmic, amino-terminal domain is necessary and sufficient for nuclear retention. When this domain is fused to a transmembrane segment of an integral membrane protein of the ER/plasma membrane, the chimeric protein is localized in the inner nuclear membrane. The transmembrane segment of emerin is not targeted to the inner nuclear membrane. Fluorescence photobleaching experiments of emerin fused to green fluorescent protein demonstrate that the diffusional mobility (D) of emerin is decreased in the inner nuclear membrane (D=0.10+/-0.01 microm2/second) compared to the ER membrane (D=0.32+/-0.01 microm2/second). This is in agreement with a model where integral proteins reach the inner nuclear membrane by lateral diffusion and are retained there by association with nucleoplasmic components. Some overexpressed emerin-green fluorescent protein also reaches the plasma membrane of transfected cells, where its diffusion is similar to that in the inner nuclear membrane, suggesting that emerin may also associate with non-nuclear structures.

Characterisation of human natural anti-sheep xenoantibodies.

Currently, the pig species is regarded as the most likely organ donor for human xenotransplantation in the future. However, it cannot be granted that the pig will be the optimal species of choice. We have studied human anti-sheep antibodies in comparison with anti-pig antibodies. The anti-sheep lymphocytotoxic and hemagglutination titers were in the range 8 to 128 and 2 to 32, respectively, in single individuals, which were considerably lower than the anti-pig titers of these individuals. Perfusion of sheep kidneys with human blood reduced the anti-sheep xenoantibody titers to zero as measured by lymphocytotoxic, hemagglutination, and sheep aortic endothelial cell antibody binding assays. The perfused kidneys showed generalised depositions of human IgM and C3c in the vascular tree and focal depositions of C1q and fibrin. Obliteration of capillaries by human platelets and polymorphonuclear cells were observed. Total neutral glycolipid fractions were isolated from sheep intestinal, pancreatic, and kidney tissues. By using a chromatogram binding assay, a monoclonal anti-Forssman antibody identified a single compound with five sugar residues in all organs. Several glycolipid bands were stained in all organs by the Gal(alpha)1-specific lectin I-B4 from Griffonia (Bandeiraea) Simplicifolia. A human AB serum pool showed staining by both IgG and IgM antibodies of the Forssman and Gal(alpha)1-terminating components as well as some other, not structurally identified, components. The Forssman and Gal(alpha)1-reactivity in human sera could be eliminated by immunoadsorption using Forssman and Gal(alpha)1-3Gal-immunoadsorbent columns, respectively. Immunostaining of sheep kidney tissue sections showed the presence of Gal(alpha)1-terminating epitopes by immunoperoxidase and immunogold silver staining techniques. Proximal convoluted tubules showed a strong staining, while thin loops of Henle, collecting ducts, urothelium, and vessels showed a weaker staining. Distal convoluted tubules and thick loops of Henle were completely negative. In summary, human serum contains anti-sheep xenoantibodies reacting mainly with the Forssman and Gal(alpha)1-determinants in sheep tissues and the anti-sheep antibody titers are lower than the corresponding anti-pig titers.

Soluble saccharides block the inhibition of agonist-induced human platelet aggregation observed after in vitro incubation of human platelet-rich plasma with porcine aortic endothelial cells.

Platelet aggregation is a prominent feature in the hyperacute process of vascularized allografts and xenografts. In a study of extracorporeal connection of pig kidneys to the blood circulation of human volunteers, we observed in one case considerable destruction of human platelets in the pig kidney without signs of hyperacute rejection or microthrombi formation. In the present study, we have investigated the agonist-induced aggregation of human platelets in mixtures with porcine aortic endothelial cells (PAEC). In vitro incubation of human platelet-rich plasma (PRP) with PAEC inhibited platelet aggregation induced by ADP, collagen and arachidonic acid in a time-dependent manner and partially inhibited adrenalin-induced aggregation. Aggregation of the human platelets could not be induced by high concentrations of ADP (20 microM) to overcome the inhibition capacity of the PAEC. The PAEC inhibiting effect could be transferred by the supernatants of PAEC/PRP and PAEC/PPP incubation mixtures. Preincubation of the PAEC with aspirin, but not with NG-methyl-L-Arg, reduced the aggregation inhibitory effect. Control experiments mixing human umbilical vein endothelial cells (HUVEC) and human PRP or mixing porcine PRP and PAEC did not elicit any inhibition of ADP-induced platelet aggregation. The aggregation inhibition effect could partially be blocked by preincubation of PRP with soluble Gal alpha 1-3Gal, Gal alpha 1-3 beta 1-4GlcNAc, lactose, galactose, and glucose, but not by lactosamine, galactosamine, or glucosamine. The Gal alpha 1-3Gal disaccharide was most effective in blocking aggregation inhibition, and to a similar extent as its ability to block the human anti-pig lymphocytotoxicity reaction. In conclusion, the data indicate that PAEC, upon stimulation by human anti-pig xenoantibodies in a nondynamic system, inhibits agonist-induced human platelet aggregation, and that this effect is probably at least partially caused by prostacyclin released from the PAEC.