Late enzymes of vindoline biosynthesis

From differentiated plants of Catharanthus roseus (L.) G. Don we have isolated a specific enzyme of the vindoline biosynthetic pathway catalysing the S-adenosylmethionine-dependent methylation of 11-O-demethyl-17-O-deacetyl-vindoline. The enzyme we named S-adenosyl-L-methionine : 11-O-demethyl-17-O-deacetylvindoline 11-O-methyltransferase. This transferase exhibits a high substrate specificity. Obviously the O-methylation at C-11 precedes the O-acetylation at the C-17 position during the biosynthesis of vindoline.A second enzyme was detected which hydrolyses the acetyl function of vindoline. The distribution of this acetylesterase in C. roseus plants demonstrates that the enzyme is not specifically associated with the vindoline distribution in the plant material. Most probably this enzyme plays no essential role in the biosynthesis of vindoline.     

Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.     

Primary production data from the south-eastern Weddell Sea

Summary Phytoplankton production for three size classes (<20 m, 20–100 m, >100 m), total primary production and qualitative composition of phytoplankton populations were recorded from 18 stations in the south-eastern Weddell Sea in February/March 1983. Total primary production ranged between 80 and 1670 mg C m^-2 d^-1 with an average of 670 mg C m^-2 d^-1, nearly 70% of which was contributed by the <20 m size fraction (usually pennate and/or centric diatoms). Production of phytoplankton was in the higher range of values reported by other authors for the same region. Variations in primary production could not be attributed to composition of populations, ambient light levels or concentrations of macronutrients (N, P, Si). Phytoplankton populations had a higher diversity in the deeper parts of the Weddell Sea and coincided with different oceanographic situations. Three zones (along the shelf-ice edge from Atka Bay to Halley Bay, west of Halley Bay and off the Filchner/Rønne Ice Shelf) with different communities could be clearly distinguished.     

Impaired natural killer cell function in hemophiliacs with or without continuous substitution

In the present study, 28 hemophiliacs substituted continuously and 5 hemophiliacs who had received almost no blood products were investigated. Cells of OKT 3+, OKT 4+, and OKT 8+ subsets were counted. Percoll separated fractions of peripheral blood mononuclear cells were examined by morphological criteria and were tested for NK cell activity. We found that the NK cell activity of both groups of hemophiliacs was decreased on testing Ficoll separated cells or low density Percoll separated cells. Normal NK cell activity was found in medium density cells of hemophiliacs. Two possible explanations are discussed: first, the NK cell activity may be suppressed in hemophiliacs and secondly, there may be a block in maturation of NK cell activity. It is unlikely that chronic substitution by blood products counts for these alterations. The possible role of chronic infections is discussed.     

2-Bromoethyl glycosides in glycoside synthesis: preparation of glycoproteins containing alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc

The applicability of 2-bromoethyl glycosides in carbohydrate synthesis is demonstrated by the synthesis of glycosides of alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc. The bromoethyl aglycon was transformed into the methoxycarbonylethylthioethyl spacer, which allowed coupling of the sugars to proteins (BSA and KLH).     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

The Electrical Conductivity of Bovine Milk in Mastitis Diagnosis

The electrical conductivity (EC) of milk is mainly a function of the electrolyte concentration in the milk and therefore raised in mastitis. The present investigation was aimed at elaborating, if possible, a diagnostic model for screening purposes based on EC determinations and consistent with the diagnostic procedures and interpretations commonly used in laboratory milk diagnosis in the Nordic countries (Klastrup 1975). According to this diagnosis (here called reference diagnosis) cell numbers above 300,000/ml (cell count or the corresponding CMT-score) in foremilk quarter samples during the main part of the lactation period and significantly above the lowest value on within-udder comparison during late lactation are considered indicative of mastitis and bacteriological examinations are made when called for.     

Expression of L3T4 molecules on Lyt-2+, class II-restricted antigen-specific T suppressor lymphocytes

Three bovine serum albumin-specific Lyt-2⁺ T suppressor (Ts) cell clones from CBA/J mice have been analyzed with regard to expression of L3T4 molecules. All three Ts-cell clones can be stained with monoclonal antibodies (mAb) to L3T4. Tested for the two clones restricted to recognition of E^k determinants, antigen-specific proliferation on antigen-presenting cells, but not the proliferation induced by conditioned medium can be inhibited by L314-specific mAb. In a similar way, Ts-cell cytolytic effector functions can be blocked by L3T4-specific mAb. Thus L3T4 structures seem to play a role in Ts-cell functions. Furthermore, the data support the view that L3T4 expression can be a property of class II-restricted T cells irrespective of their Lyt phenotype.     

Electron microscopic cytochemical localization ofα-hydroxyacid oxidase in rat liver

Summary The substrate specificity and the intraperoxisomal localization of -hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37°C in a medium containing 3 mM CeCl₃, 100 mM NaN₃ and 5 mM of an -hydroxyacid in 0.1M of one of the following buffers: Pipes, Mops, Na-cacodylate,Tris-maleate, all adjusted to pH 7.8. Ten different -hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic andl--isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in theTris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections inTris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of -hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.Abbreviations -HAOX l-hydroxyacid oxidase - Argininic acid l--hydroxy--guanidinovaleric acid - Pipes piperazine-N,N-bis(2ethane sulfonic acid) - Mops 3(N-morpholino) propane sulfonic acid - Tris tris-(hydroxymethyl)-aminomethane - Luminol 5-amino-2,3 dihydrophthalazine-1,4-dione - GA glutaraldehyde