Studies of platelets with heavy metal impregnation techniques

Summary Vaious methods of heavy metal impregnations were performed on human platelets. The optimal technique consisted of glutaraldehyde fixation, incubation in warm uranyl acetate at a pH of 3.5, followed by a double solution of lead and copper, and finally overnight immersion in cold osmium tetroxide. Semi-thin sections, viewed at 90 kV, revealed three types of platelets: (1) reticular cells, with a prominent tubular network and very dark granules in a pale cytoplasm; (2) dark cells, with an electron-dense cytoplasm; and (3) pale cells, with microvesicles and non-staining granules. Pre-treatments with EGTA, aspirin and various platelet activators altered the appearances and proportions of the three cell types.A cell-partitioning two-phase polymer system showed that the sub-grouping is related to surface membrane properties, the cells retained in the top phase being exclusively type 2 dark cells.The changes in cell type distribution produced by activation show that metal impregnation may be a useful method for studying structure-function correlations in platelets.     

Measurement of the protein backbone dihedral angle φ based on quantification of remote CSA/DD interference in inter-residue 13C′(i − 1)-13Cα(i) multiple-quantum coherences

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between ¹H(i)-¹³C(i) dipolar and ¹³C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of ¹⁵N magnetization to the inter- and intra-residue ¹³C carbons as well as the directly attached carbonyl carbon ¹³C. Results obtained on ¹³C,¹⁵N-labeled ubiquitin demonstrate the potential of the method.     

The influence of anther cold pretreatments and donor plant genotypes on in vitro androgenesis in wheat (Triticum aestivum L.)

Cold pretreatments applied to excised anthers in liquid potato 2 medium proved to be unnecessary. Generally, cold pretreatments inhibited anther response and productivity as the duration was lengthened or as the pretreatment temperature was lowered. There were significant differences in response attributed to the anther donor genotype. Green and albino plants as well as roots only have been regenerated from the spring wheat cultivars Sinton, Neepawa, Pitic 62 and DW 50. Most plants were haploid.     

Secretion of lysosomal enzymes by human placental villi in vitro

Summary Chorionic villi from first trimester and term human placentas have been incubated in vitro and shown to release the lysosomal enzymes, -hexosaminidase, -glucosidase and -gluctlronidase. There was negligible release of the cytoplasmic enzyme, lactate dehydrogenase, under the same conditions. The first trimester villi released proportionally more of their lysosomal enzyme content than did the term villi. Extracellular levels of -hexosaminidase were raised and those of -glucosidase and, -glucuronidase were lowered when tissue was incubated with 1 M colchicine, suggesting that microtubules are involved in the control of lysosomal enzyme release from placental villi.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, Wichita and Cheyenne. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in Wichita than in Cheyenne. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between Wichita and Cheyenne. While the allele on Wichita chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on Cheyenne chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in Wichita. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.This work was supported in part by USDA-Competitive Research Grants Office grant No. 90-37140-5426 to P.S.B. Contribution from Agricultural Research Division, University of Nebraska. Journal Series Number 10304     

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes

Synopsis Fixation under improper conditions ofin vitro cultivated cells results in an extensive diffusion of the lysosomal enzyme acid phosphatase because of the influence of a low effective osmotic pressure. In the present investigation, advantage was taken of this predictable diffusion in order to establish whether or not leakage of acid phosphatase could take place through ultrastructurally intact lysosomal membranes.In order to reveal small holes in the lysosomal membranes, secondary lysosomes were labelled with thorium dioxide particles, which were presumed to appear free in the cell sap if ruptures in the membranes larger than about Ioo Å were created.The experiments revealed that following the fixation ofin vitro cultivated human glia cells under improper conditions, mitochondria and ground cytoplasm show considerable swelling artifacts, while secondary lysosomes appear to be essentially unaffected. The lysosomes, nevertheless, apparently lost most of their content of acid phosphatase, as judged from enzyme cytochemical studies. These findings indicate that leakage of acid phosphatase from ultrastructurally intact lysosomes is possible.     

The linkage of Hb Valletta [α2β287(F3)Thr→Pro] and Hb F-Malta-I [α2 Gγ2117(G19)His→Arg] in the Maltese population

Summary We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a ThrPro substitution at position 87 of the chain. This mutation was found to be linked to that of the chain variant Hb F-Malta-I with a HisArg mutation at position 117 of the ^G chain. Both variants were detected in the blood samples of 34 Maltese and two Italian new-born babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 -thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the ^G-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the ^G- and -globin genes, 27–28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency.     

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle

Summary Whith the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of andocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme was not 5-nucleotidase for a typical lysosomal acid phosphatase. The T-tubular enzyme hydrolysed glucose 6-phosphate and -glycerophosphate at pH 5.0 but not cytidine-5-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a mique phosphatase present on mucle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5-nucleotidase.     

Relations between Computational and Experimental Engineering Approaches to Molecules from Molecular Fragments

Computational techniques have become important tools in the preliminary stages of the design of new molecules. The mutual arrangements of interacting molecular parts and the required accessibility of important reactive groups on the peripheral regions of possible new molecules can be tested by computational means before the expensive and often complex synthetic methods are used to the actual construction of these molecules. There are some common features involved in the computational representation of molecular fragments and the synthetic methodologies used in the process of incorporating actual molecular fragments in such engineered molecules. One trend that appears to link these two approaches is based on the following observation: the greater local autonomy is shown by the calculated electron density contribution of a given molecular fragment, the more likely that this fragment can be regarded as a suitable building block of the molecule, and it is also more likely that there are convenient synthetic methods for the delivery of this fragment to its desired target location in the new molecule to be synthesized. For a precise formulation of this statement, a new concept, the degree of molecular fragment autonomy is introduced, using the inherent properties of molecular electron densities.     

Regional mapping of the human gene for lysosomal α-glucosidase by in situ hybridization

Summary The current approach to the chromosomal localization of genes coding for lysosomal enzymes has been the correlation of enzymatic and karyotypic analyses of human-rodent somatic cell hybrids. The feasibility of regional mapping depends on the availability of human cells with informative chromosomal rearrangements. In this communication we report the first localization of a gene coding for a lysosomal enzyme by in situ hybridization. The application of an acid -glucosidase cDNA probe to normal human chromosomes allowed direct regional mapping of the -glucosidase locus (GAA) to the region q23q25 of chromosome 17.     

Lysosomal α-galactosidase in endothelial cell cultures established from a fabry hemizygous and normal umbilical veins

Summary An endothelial cell line has been established from the umbilical vein obtained after abortion of a male fetus suffering from Fabry disease. This X-linked inborn error of glycosphingolipid catabolism results from deficiency of the lysosomal hydrolase -galactosidase. A the clinical manifestations of the disease are mainly caused by glycosphingolipid depositions in the endothelium of all vessels. The hemizygous cell line and eight endothelial cell lines originating from the umblical cords of normal newborns were grown for more than 10 passages. They had a short generation time that allowed us to get sufficient cells for qualitative and quantive investigations of -galactosidase. The enzyme in normal endothelial cells had a similar thermostability and isoelectric focusing pattern as that in fibroblasts, but the activity was essentially higher in endothelial cells. The hemizygous endothelial cells were deficient in -galactosidase A. It is concluded that endothelial cell lines are an important alternative to fibroblasts for in vitro studies of the lysosomal storage diseases.     

Produktivität und Energiegehalte von Gefä\pflanzen im Adventdalen (Spitzbergen)

Zusammenfassung Im Sommer 1973 wurden im Adventdalen/Spitzbergen (=78°15 N, =17° E bis 15°30 E) pflanzenökologische Untersuchungen gemacht. Im einzelnen wurden während 13 Tagen Temperaturen und Strahlung gemessen. Ferner wurden Pflanzen und Pflanzengesellschaften nach der Erntemethode auf ihre Biomasse und Produktivität untersucht. Der Kaloriengehalt der Pflanzen wurde später im Labor ermittelt.Gegenüber anderen arktischen Gebieten ergaben sich höhere Produktivitätswerte, vor allem für gräser und grasartige Pflanzen. Dies ist zurückzuführen auf die klimatische Sonderstellung des Untersuchungsgebietes.

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Summary In the summer of 1973 in Adventdalen, spitzbergen (=78°15 lat., =17° to 15°30 long.) ecological studies of vascular plants were done. In particular, temperatures and radiation were measured over a 13-day period. In addition, plants and groups of plants were investigated according to the harvest method with respect to their biomasses and productivity. The caloric content of the plants was subsequently determined in the laboratory.In relation to other arctic areas, the area under study yielded higher productivity values, especially for grasses and grasslike plants. This is attributed to the special climatic position of the area.

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Unterstützt durch die Deutsche Forschungsgemeinschaft.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

On the role of enzyme kinetic parameters in determining the effectiveness with which channelling can decrease the size of a metabolite pool

Recently, it has been argued that the phenomenon of direct transfer of intermediate metabolites between adjacent enzymes, also known as metabolic channelling, would not decrease the concentration of those intermediates in the bulk solution. However, this conclusion has been drawn by extrapolation from the results of simulations with a rather restricted set of parameters. We show that, for a number of kinetic cases, the existence of metabolic channelling can decrease the size of the soluble pool of intermediates. When the enzyme(s) downstream of the channel have a catalytic capacity that is large relative to the enzymes upstream of the channel, the decrease of concentration can be substantial (3 orders of magnitude).     

Histochemistry of myelin. IX. Neutral and acid proteinases in early Wallerian degeneration

Synopsis Acid and neutral proteinases, leucine aminopeptidase (l-leucyl--naphthylamidase) and acid phosphatase were studied in rat sciatic nerves undergoing Wallerian degeneration. Biochemical evidence indicated that increased activity of both proteases and acid phosphatase occurred by 12 hr after nerve section. Histochemical changes in these three enzymes were apparent after three days. Biochemical estimation of neutral leucine aminopeptidase (an enzyme predominantly located in myelin in the normal peripheral nerve) showed increased activity near the of the first week of degeneration. During the second week after nerve section all the enzymes studied became markedly more active. The parallel increase in activity of acid proteinase and acid phosphatase and the similarities in their histochemical distribution suggest that the acid proteinase is of lysosomal origin. Such changes in early Wallerian degeneration appear to precede macrophage invasion of the nerve and to arise mainly from the degenerating axon, the Schwann cell, or both. In spite of the delayed increase in leucine aminopeptidase it seems possible that some proteinase activity also arises from myelin.Research Associate supported by the British Multiple Sclerosis Society     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.