Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

Isolation and characterization of neurokinin A, neurokinin A(3-10) and neurokinin A(4-10) from a neutral water extract of a metastatic ileal carcinoid tumour

A metastasis to the right liver lobe of an argyrophil/argentaffin midgut carcinoid tumour in a patient with the classical carcinoid syndrome was examined for the presence of tachykinins other than substance P, using a specific antiserum. The extract was initially purified using SepPak cartridges, and subsequently subjected to cation-exchange chromatography on SP Sephadex C-25 which separated the immunoreactive material into two main components (components I and II). Both were further purified by anion-exchange chromatography on DEAE-Sephadex A-25, and by reverse-phase fast protein liquid chromatography. Component II was identified as neurokinin A by its immunochemical and chromatographic properties and amino acid sequence analysis. Component I consisted of two molecular forms which were identified as neurokinin A(3-10) and neurokinin A(4-10) by amino acid sequence analysis. The tumour tissue contained only small amounts of the eledoisin-like peptide that has earlier been demonstrated in mammalian tissues. Although this component behaved like the nonmammalian peptide eledoisin on reverse-phase HPLC and on reverse-phase ion-pair chromatography, eledoisin-specific antiserum E2 indicated that eledoisin-like peptide is not identical to eledoisin. Neurokinin A in carcinoid tumours has an N-terminal heterogeneity; this multiplicity constitutes a further support for the hypothesis that carcinoid tumours produce a number of tachykinins which may be present in different relative amounts in individual patients and may contribute to the individual differences in symptomatology.     

Glycolipid- and glycoprotein-based blood group A antigen expression in human thrombocytes. A1/A2 difference

Total non-acid glycolipid fractions and total sodium dodecylsulphate (SDS) solubilized protein fractions were isolated from human thrombocytes obtained from single human donors having different blood group A₁/A₂ phenotypes. The blood group A glycolipid antigens were characterized by immunostaining of thin layer plates with different monoclonal anti-A antibodies. The glycoproteins carrying blood group A epitopes were identified by SDS-PAGE and Western blot analysis using a monoclonal anti-A antibody. Blood group A glycolipid antigens were found in both A₁ and A₂ thrombocytes but the A₂ individuals expressed at least ten times less A glycolipids compared to the A₁ individuals. Expression of A type 3/4 chain and small amounts of A type 1 chain glycolipids were seen in thrombocytes of both A₁ and A₂ individuals, while the type 2 chain A glycolipids appeared to be missing from the A₂ thrombocytes. Blood group A reactive glycoproteins were only found in thrombocytes of A₁ individuals and could not be detected in A₂ individuals or a blood group O individual. The major blood group A glycoprotein were found as a double band migrating in the 130 kDa region.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPTLC high performance thin layer chromatography - CBB Coomassie brilliant blue - GVH graft versus host Part of this work was presented at the Xth International Symposium on Glycoconjugates, Jerusalem, Israel. September, 1989.In the short hand designation for glycolipids, the letter indicate blood group determinant, the first numeral, the number of sugar residues, and the second numeral, the type of carbohydrate chain. Thus, A-6-1 means a hexaglycosylceramide with a blood group A determinant based on the type 1 carbohydrate chain.     

The Influence of Feeding and Oral Rehydration on the Bioavailability of Oxytetracycline in Calves

The influence of feeding on the bioavailability of oxytetracycline was studied in preruminant calves. Oxytetracycline was given in water as a drench to fasting calves or was mixed in the milk replacer. Compared to water the bioavailability was significantly reduced (53.5%) when oxytetracycline was mixed in the milk re-placer. A further reduction, 83.3 %, occurred when the calves were treated one hour post milk feeding. Also concentrate was found to reduce the bioavailability. Very high serum levels were recorded when the drug was given in an oral rehydration solution, pH 4.9, containing glycine. The values obtained when an alkaline (pH 8.3) solution without glycine was used did not differ from the levels recorded when oxytetracycline was given in water. It was suggested that the use of oxytetra-cyclines in feeds may be questioned because of their well-known complex forming ability.     

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Altered Growth and Wood Characteristics in Transgenic Hybrid Aspen Expressing Agrobacterium tumefaciens T-DNA Indoleacetic Acid-Biosynthetic Genes

A key regulator of cambial growth is the plant hormone indoleacetic acid (IAA). Here we report on altered wood characteristics and growth patterns in transgenic hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) expressing Agrobacterium tumefaciens T-DNA IAA-biosynthetic iaaM and iaaH genes. Eighteen lines simultaneously expressing both genes were regenerated. Of these, four lines, verified to be transgenic by northern blot analysis, were selected and raised under controlled growth conditions. All four lines were affected in their growth patterns, including alterations in height and stem diameter growth, internode elongation, leaf enlargement, and degree of apical dominance. Two transgenic lines, showing the most distinct phenotypic deviation from the wild type, were characterized in more detail for free and conjugated IAA levels and for wood characteristics. Both lines showed an altered IAA balance, particularly in mature leaves and roots where IAA levels were elevated. They also exhibited changes in wood anatomy, most notably a reduction in vessel size, an increase in vessel density, and changes in ray development. Thus, the recent development of techniques for gene transfer to forest trees enabled us to investigate the influence of an altered IAA balance on xylem development in an intact experimental system. In addition, the results demonstrate the possibility of manipulating wood properties in a forest tree through controlled changes of IAA concentration and distribution.     

Visna Virus Meningoencephalomyelitis in Goats

A progressive paresis was encountered in herds of Swedish goats. The symptoms developed during a period of weeks or months, and were initially often seen as a weakness of the hind limbs before the animals became paralytic. The development and the histopathological lesions of the disease in the GNS and the lungs were similar to those of visna in sheep. In vitro grown choroid plexus cells, prepared from affected goats, showed foci of polykaryocytes. Electron microscopy revealed the presence of particles morphologically similar to those of sheep visna virus (SVV). Goats experimentally infected with the goat visna virus (GVV) developed GNS lesions similar to those of visna in sheep and became seropositive to SVV. The results of complement fixation tests, carried out on sera from 11 goat herds, showed a coincidence between seropositiveness and the occurrence of disease in one and the same herd. Using the ELISA method, an average of 80 % of the goats in 5 herds were found to be seropositive to GVV.     

Informed consent: study of quality of information given to participants in a clinical trial.

OBJECTIVE--To determine whether the participants in a clinical trial had perceived adequate information about the trial according to the guidelines of the Declaration of Helsinki. DESIGN--About 18 months after the end of a gynaecological clinical trial the participants received a questionnaire by post, which focused on the quality of the information given to them before entering the trial. Neither researchers nor participants were aware in advance that the trial would become the subject of this follow up investigation. SETTING--Eight different centres in Sweden. SUBJECTS--43 women out of the 53 who completed the trial (mean (range) age 23 (16 to 35) years) returned the questionnaire. MAIN OUTCOME MEASURES--Adequacy of the information (based on requirements of the Declaration of Helsinki) to enable the following: understanding of the aims of the study; awareness of what participation meant; and awareness of the possibility of withdrawing from participation at any time. Motives for agreeing to participate, and a subjective evaluation of the given information were also recorded. RESULTS--All but one of the participants had been aware that they were taking part in a research project. Five women stated that they had not been aware that a second laparoscopy was performed only for research reasons. Seven women reported that they had not been aware of the meaning of participating in the project and 17 that they had had no information about the possibility of withdrawing from the study whenever they wanted. In the subjective rating 22 women considered the information given as good or very good. There was a systematic variation in the quality of the given information among the eight centres. CONCLUSION--Although all but one of the participants had been aware that they were taking part in a clinical trial, the quality of the information understood and recalled by participants varied, and in many cases clearly did not meet the guidelines of the Declaration of Helsinki. Variations among centres in participants'' perception of information suggest that deficiencies in perception may be caused by informers rather than the participants.