Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

Degradation of microvascular brain endothelial cell beta-catenin after co-culture with activated neutrophils from patients undergoing cardiac surgery with prolonged cardiopulmonary bypass

The adhesion of highly activated neutrophils to cerebral microvascular endothelial cells (MVECs) may contribute to disruption and hyperpermeability of the blood-brain barrier (BBB) after cardiac surgery with prolonged cardiopulmonary bypass (CPB). A correlation between CPB duration and neutrophil-mediated BBB damage has not been investigated on the cellular level yet. Therefore, we studied the effects of neutrophils from cardiac surgery patients with CPB time <80 min (group I; n=8) and >80 min (group II; n=8) on the integrity of cultured porcine MVEC. Ex vivo, neutrophils of group II but not of group I significantly degraded the zonula adherens molecule beta-catenin whereas VE-cadherin and occludin were not modified. The transendothelial electric resistance as a measure for the integrity of the endothelial monolayers was reduced over time in both groups. In conclusion, prolonged CPB time entails neutrophil-mediated decrease in MVEC beta-catenin expression, and thus may be an important trigger for BBB disruption.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits approximately 2000-fold lower K(m) values for NADP(+) and NADPH than for NAD(+) and NADH and recognizes both retinoids and lipid peroxidation products (C(9) aldehydes) as substrates. The k(cat) values of RDH12 for retinaldehydes and C(9) aldehydes are similar, but the K(m) values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k(cat)/K(m) approximately 900 min(-)(1) microM(-)(1)), followed by 11-cis-retinal (450 min(-)(1) mM(-)(1)) and 9-cis-retinal (100 min(-)(1) mM(-)(1)). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.     

Prediction of collagen stability from amino acid sequence

An algorithm was derived to relate the amino acid sequence of a collagen triple helix to its thermal stability. This calculation is based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm. However, predicted T(m) values are significantly higher than experimental values when there are strings of oppositely charged residues or concentrations of like charges near the terminus. Application of the algorithm to collagen sequences highlights regions of unusually high or low stability, and these regions often correlate with biologically significant features. The prediction of stability from sequence indicates an understanding of the major forces maintaining this protein motif. The use of highly favorable KGE and KGD sequences is seen to complement the stabilizing effects of imino acids in modulating stability and may become dominant in the collagenous domains of bacterial proteins that lack hydroxyproline. The effect of single amino acid mutations in the X and Y positions can be evaluated with this algorithm. An interactive collagen stability calculator based on this algorithm is available online.     

Cellular uptake of mammalian heparanase precursor involves low density lipoprotein receptor-related proteins, mannose 6-phosphate receptors, and heparan sulfate proteoglycans

Mammalian heparanase, strongly implicated in the regulation of cell growth, migration, and differentiation, plays a crucial role in inflammation, angiogenesis, and metastasis. There is thus a clear need for understanding how heparanase activity is regulated. Cells can generate an active form of the enzyme from a larger inactive precursor protein by a process of secretion-recapture, internalization, and proteolytic processing in late endosomes/lysosomes. Cell surface heparan sulfate proteoglycans are the sole known components with a role in this trafficking of the heparanase precursor. Here, we provide evidence that heparan sulfate proteoglycans are not strictly required for this process. More importantly, by heparanase transfection, binding, and uptake experiments and by using a combination of specific inhibitors and receptor-defective cells, we have identified low density lipoprotein receptor-related proteins and mannose 6-phosphate receptors as key elements of the receptor system that mediates the capture of secreted heparanase precursor and its trafficking to the intracellular site of processing/activation.     

Systematics ofPlagiochila sect.Glaucescentes Carl (Hepaticae) from tropical America: A morphological and chemotaxonomical approach

The systematics of theGlaucescentes Carl, a poorly known neotropical section of the large genusPlagiochila, is revised based on a large set of gametophytic, sporophytic and chemical characters. Three species are recognized,P. buchtiniana Steph.,P. longispina Lindenb. and Gottsche andP. diversifolia Lindenb. and Gottsche, the latter with two chemotypes. Twelve binomina are reduced to synonymy. The species occur in montane regions of tropical America, especially in the northern and central Andes. In addition,P. longispina is newly reported from the Azores, where it was previously known asP. allorgei Herzog and Perss. TheGlaucescentes are excellently characterized by morphological and chemical features and are clearly separated from theContiguae Carl, a group often confused with theGlaucescentes. Morphologically, theGlaucescentes stand apart by the brown oil bodies, thinwalled inner capsule wall cells and partly unequally bispiral elaters. Chemically, they are characterized by the accumulation of partially hydrogenated bibenzylderivatives (longispinone, longispinol), 3-benzylphthalides and various flavonoids, and by the low production of terpenoids.Publication No. 142 of the Arbeitskreis Chemie und Biologie der Moose, Universität des Saarlandes, Saarbrücken.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Wirkstoffe der B-Gruppe in der Bienennahrung

Summary Through a test (Tribolium-test) which was developed at our institute byK. Offhaus andG. Fröbrich, pollen of ca. 35 and antheres of almost 70 different plants, as well as several honey-proofs and food-sap for the queen could be examined in regard to their effectiveness. The following B-vitamins were tested, in them: B1, B2, B6, -Biotin, Pantothenic Acid, Fol Acid, Nicotin amid, Cholinchlorid, B12 (in honey only!) and the imago-factor.Pollen dispensers, which have been wellknown to the bee-keeper for a long time as a good feeding-ground for the bees (Crocus, Salix, Salvia, Galanthus, etc.), are the best sources for all B-vitamins. First of all among the effectives of the B-group the beekeeper must be interested in the vitamins Pantothenic Acid and -Biotin. They are present in great quantity in the food-sap for the queen and, without doubt, derive from the pollen-food of those bees associated with queen. For the problem of determination, Pantothenic Acid and -Biotin (?) probably play the decisive role. The best sources of Pantothenic Acid for the bees are amongst those plants examined by us: sage, robinia, rose, and lysimachia; and amongst the anemophiles: scleranth-grass and wheat. Then follow the spring-flowers: Crocus, scilla, gagea, narcissus, salix, and among the later flourishing plants: horse-chestnut, iris, nothera, campanula rapuncoloides. Yet 1/4 of all tested plants have pollen with a small percentage of Pantothenic Acid. An especially large quantity of Biotin is found in the antheres of salix and campanula-types. Yet also galanthus, scilla, crocus, gagea, primula, and apple-tree have antheres which contain a great deal of Biotin. In some pollen, respectively antheres, a new factor, necessary for the metamorphosis of Tribolium (and, perhaps, for all other insects too?), was identified, which has also vitamin, character (TIF byFröbrich-Offhaus,Bt byFraenkel).Poisonous for our test subjects were the antheres of philadelphus, digitalis (pollen not poisonous!) and Colchicum. The pollen of the meadow-saffron produces great losses, yet a few insects develop nevertheless, so that it is in question whether this pollen is poisonous.Honey, however, contains only a very small quantity of B-vitamins and those small traces of vitamin probably derive from the pollen which is always mixed with the honey. Folic Acid and Pyridoxin are of the highest percentage, -Biotin and Cholinchlorid follow at a distance—nicotinacid is merely found in traits. Completely negative results were obtained in tests for B₁, B₂, B12 and Pantothenacid.

---

Résumé Au moyen d'un test mis au point à notre institut parK. Offhaus etG. Fröbrich, on a pu examiner le spectre d'efficacité du pollen d'environ 35 et des anthères de presque 70 plantes différentes ainsi que celui d'échantillons de miel et de suc nourricier des reines. On y releva les vitamines B suivantes: B₁, B₂, B₆, Biotine , acide pantothénique, acide folique, amide d'acide nicotinique, chloriede-cholin, B₁₂ (seulement dans le miel) et le facteur Imago.Les fournisseurs de pollen, bien connus depuis longtemps de l'apiculteur comme bon terrain de pâture pour les abeilles (Crocus, Salix, Salvia, Galanthus), constituent les sources les meilleures de toutes les vitamines B₁. Parmi les excitants du groupe B, la vitamine acide pantothénique et biotine intéresseront en premier lieu l'apiculteur. On les trouve en quantités particulièrement importantes dans le suc nourricier de la reine, et elles proviennent sans aucun doute de la nourriture faite de pollen des abeilles nourricières. Dans le problème de la détermination, on suppose qu'elles jouent un rôle décisif. Les meilleures sources d'acide pantothénique pour les abeilles parmi les plantes que nous avons examinées à cet égard, sont: salvia, robinier, rose lysimachia, et parmi les anémophiles: scléranthe et froment. Parmi les fleurs de printemps, ce sont:crocus, scilla, gagea, narcissus, salix; parmi les fleurs dont la floraison est plus tardive:aesculus, iris, nothera, campanules rapuncoloides. Le quart des plantes examinées out un pollen avec faible pourcentage d'acide pantothénique. Les anthères desSalix et des différentes variétés de Campanules sont particulièrement riches en biotine. De même, les anthères desgalanthus, scilla, crocus, gagea, primula et pommiers sont relativement riches en biotine. Dans certains pollens, voire même certaines anthères, on a pu prouver l'existence d'un nouveau facteur indispensable pour la métamorphose duTribolium (et peut-être même pour celle de tous les insectes) et présentant par conséquent un caractère vitaminique (TIF chezFröbrich-Offhaus, B_t chezFraenkel).Ont réagi de façon vénéneuse sur la matière du test les anthères duPhiladelphus, digitalis (son pollen est inoffensif), de l'anthirhinum (son pollen est également inoffensif) et ducolchicum. Le pollen du colchique entraîne de nombreux manques; pourtant certains insectes, quoiqu'en petit nombre, se développent, de sorte que la question se pose si ce pollen est vénéneux on non.Par contre, le miel est pauvre en vitamine B et les quelques traces de vitamines que l'on peut relever proviennent vraisemblablement du pollen qui se mêle toujours au miel. L'acide folique et le Pyrodoxin sont encore largement représentés, ensuite viennent la biotine et le chloride cholin; on ne, relève l'amide d'acide nicotinique qu'à l'état de traces. Les expériences recherchant l'existence de B₁, B₂, B₁₂ et d'acide pantothénique se sont montrées totalement négatives.

---

---

Unserm verehrten Lehrer Herrn Prof. Dr. Paul Buchner, zu seinem 70. Geburtstag am 12. April 1956 gewidmet.