Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

Biomechanical measurements on scaphoid bone screws in an experimental model

A number of screws commonly used for internal fixation in scaphoid bone fractures and nonunions are compared regarding biomechanical properties and clinical applicability. The experiments were carried out on models made of ash-wood, representing a reconstruction and fixation as is performed in a cortico-cancellous inlay bone graft for scaphoid non-union. For fixation use was made of 2.7 and 3.5 AO/ASIF cortical screws respectively, 4.0 AO/ASIF cancellous screws, Herbert screws, and a newly designed screw called the three components screw (D.K.S.). The models with implanted screws were tested for bending strength, tensile strength and torsion stability. No large differences between the various screws were found regarding the measured parameters, so that a small intra-osteal implant such as the Herbert screw and the D.K.S., which can be inserted easily and which gives a certain amount of interfragmentary compression, will be sufficient for osteosynthesis of the scaphoid bone. In case an intra-osteal implant is not available a single 3.5 AO/ASIF cortical screw, inserted following lag-screw principles, is recommended.     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.     

Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.     

Blood–Brain Barrier Permeability to Sodium. Modification by Glucose or Insulin?

In order to explore the pathogenetic mechanism underlying the changes in blood-brain barrier sodium transport in experimental diabetes, the effects of hyperglycemia and of hypoinsulinemia were studied in nondiabetic rats. In untreated diabetes, the neocortical blood-brain barrier permeability for sodium decreased by 20% (5.6 +/- 0.7 versus 7.0 +/- 0.8 X 10(5) ml/g/s) as compared to controls. Intravenous infusion of 50% glucose for 2 h was associated with a decrease in the blood-brain barrier permeability to sodium (5.4 +/- 1.2 X 10(5) ml/g/s), whereas rats treated with an inhibitor of insulin-secretion (SMS 201-995, a somatostatin-analogue) had normal sodium permeability (7.3 +/- 2.0 X 10(5) ml/g/s). Acute insulin treatment of diabetic rats normalized the sodium permeability within a few hours as compared to a separate control group (7.7 +/- 1.1 versus 6.9 +/- 1.4 X 10(5) ml/g/s). To elucidate whether the abnormal blood-brain barrier passage is caused by a metabolic effect of glucose or by the concomitant hyperosmolality, rats were made hyperosmolar by intravenous injection of 50% mannitol. Although not statistically significant, blood-brain barrier sodium permeability increased in hyperosmolar rats as compared to the control rats (8.3 +/- 1.0 and 7.0 +/- 1.9 X 10(5) ml/g/s, respectively). It is concluded that either hyperglycemia per se or a glucose metabolite is responsible for the blood-brain barrier abnormality which occurs in diabetes. Further, we suggest that the specific decrease of sodium permeability could be the result of glucose-mediated inhibition of the Na+K+-ATPase localized at the blood-brain barrier.     

FECAL METHANOGENS AND VERTEBRATE EVOLUTION

It has been assumed that the feeding habits of vertebrates predispose the variety of intestinal differentiations and the composition of the microbial biota living in their intestinal tracts. Consequently, the presence of methanogenic bacteria in the various differentiations of the large intestine and the foregut of herbivorous vertebrates had been attributed primarily to the existence of anaerobic habitats and the availability of carbon dioxide and hydrogen originating from the fermentative microbial digestion of plant-based diets. However, Australian ratites, many murids, and several New World primates lack methanogens, despite their intestinal differentiations and their vegetarian feeding habits. Crocodiles, giant snakes, aardvarks, and ant-eaters on the other hand release significant amounts of methane. A determination of methane emissions by 253 vertebrate species confirmed that competence for intestinal methanogenic bacteria is shared by related species and higher taxa, irrespective of different feeding habits. In “methanogenic” branches of the evolutionary tree, a variety of differentiations of the large intestine evolved and, in some cases, differentiations of the foregut. In contrast, the lack of competence for methanogens in chiropterans/insectivores and carnivores apparently has precluded the evolution of specialized fermenting differentiations of the digestive tract. Our observations reveal that the presence of intestinal methanogenic bacteria is under phylogenetic rather than dietary control: competence for intestinal methanogenic bacteria is a plesiomorphic (primitive-shared) character among reptiles, birds, and mammals. This competence for methanogenic bacteria has been crucial for the evolution of the amniotes.     

Transgenic Analysis of a PotentialHoxd-11Limb Regulatory Element Present in Tetrapods and Fish

Genes of theHoxDcomplex related to theDrosophila Abd-Bgene are involved in the morphogenesis of vertebrate paired appendages.Hoxd-11,for instance, is necessary in combination with otherHoxgenes for the proper development of different parts of the tetrapod limbs. Sequence comparisons between the mouse, chicken, and zebrafishHoxd-11loci have revealed the conservation of several blocks of DNA sequence which may be of importance for the regulation ofHoxd-11expression. We have used transgenic mice to show that one of these conserved elements specifically drives expression in a proximal-posterior part of developing forelimbs. Production of mice transgenic for a full fishHoxd-11construct as well as for mouse–fishHoxd-11chimeric constructs shows that the fish counterpart of this sequence is able to elicit expression in mouse forelimbs as well, though in a slightly different domain. However, this fish element requires the presence of the mouse promoter and does not work in its own context. These results are discussed in light of both the control ofHoxdgene expression during limb development and the use of a comparative interspecies approach to understand the regulation of genes involved in vertebrate development.     

Silicone-stimulated anthraquinone production and release by Morinda citrifolia in a two-liquid-phase system

Summary A 2.5-fold increase in the release of intracellular anthraquinones was obtained by adding 1 ml L^-1 silicone A to suspension cultures of Morinda citrifolia. Cell growth and secondary metabolite production were not affected even at high silicone A concentrations. Performance of the silicone treatment in a two-liquid-phase system (5 ml n-hexadecane/50 ml medium) resulted in a 150% increase of the overall secondary metabolite productivity.     

Biotransformation of monoterpenes and sesquiterpenes by cell suspension cultures of Achillea millefolium L. ssp. millefolium

The transformation capacity of Achillea millefolium L. ssp. millefolium (yarrow) cell suspension cultures was investigated using geraniol (50mg/l) and borneol, menthol, thymol and farnesols (25mg/l) as substrates. Apart from converting these substrates into several biotransformation products, the cell suspension cultures were also able to glycosylate both the substrates and the biotransformation products. aa]Key Words bb]Achillea millefolium L. ssp. millefolium bb]Yarrow bb]Compositae bb]Biotransformation bb]Glycosylation bb]Geraniol bb]Borneol bb]Menthol bb]Thymol bb]Farnesols     

Somatic embryogenesis and organogenesis in Encephalartos dyerianus and E. natalensis

Callus cultures were initiated from zygotic embryos of Encephalartos dyerianus and E. natalensis. Callus of both species were transferred onto a modified B5 medium containing different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Somatic embryogenesis and shoot organogenesis occurred in both species. The embryos were dicotyledonary. To date none of the embryos have matured.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid