Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

Deglycosylation of a lectin intermediate during assembly of ConA

Summary Metabolic labelling of immature jackbeans (Canavalia ensiformis) has been used in a pulse-chase study to determine changes in the glycosylation pattern of polypeptides during the assembly of Concanavalin A. In an analysis that allowed the identification of 7 intermediates, only the first precursor form of the lectin was labelled with D-[U-¹⁴C]-glucosamine. These results indicate that processing of the lectin involves a novel deglycosylation event in which an N-linked oligosaccharide is removed from a protein in the absence of proteolysis.Abbreviations endo H endo -N-acetylglucosaminidase H - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - ConA Concanavalin A     

Ventilation inhomogeneity during controlled ventilation. Which index should be used?

Six indexes for diagnosing uneven ventilation by tracer gas washout were studied. The indexes were lung clearance index, mixing ratio, Becklake index, multiple-breath alveolar mixing inefficiency, moment ratio, and pulmonary clearance delay, all of which increase with impaired pulmonary gas mixing. In model lung tests, indexes that compared the actual washout curve with a calculated ideal curve (mixing ratio, multiple-breath alveolar mixing inefficiency, and pulmonary clearance delay) were unaffected by changes in tidal volume and series dead space, whereas the others varied markedly. In both spontaneously breathing and mechanically ventilated patients all indexes showed a significant difference between smokers and nonsmokers (P less than 0.002), but the indexes were somewhat different in their assessment of different ventilatory patterns. However, the mean value for all indexes, with the exception of mixing ratio, was smallest with a fast insufflation followed by an end-inspiratory pause. Any of the indexes may be useful if its limitations are recognized, but mixing ratio, multiple-breath alveolar mixing inefficiency, and pulmonary clearance delay seem preferable, because they are not affected by changes in tidal volume and dead space fraction.     

Detection of proopiomelanocortin mRNA by in situ hybridization,using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry

Summary A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (MSH/ACTH[4-11]), ws synthesized and labelled in the 3-end by use of terminal transferase. Probes tailed with either [³H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.     

Mutation analysis and prenatal diagnosis in a Lesch-Nyhan family showing non-random X-inactivation interfering with carrier detection tests

Summary A nonsense mutation at the CpG-site in the codon for Arg(169) in the gene for hypoxanthine phosphoribosyltransferase (hprt) was identified by genomic polymerase chain reaction (PCR) and DNA sequencing in cultured fibroblasts from two brothers with Lesch Nyhan's syndrome. The recurrence of mutation at this CpG-site in several unrelated Lesch-Nyhan families suggests that deamination of 5-methylcytosine is a possible mechanism for mutagenesis. The level of hprt-mRNA in the fibroblasts of the patients was similar to that in healthy controls, whereas hprt-enzyme activity was not detectable. The mutation in this family was also identified in five female relatives and prenatally in a male fetus. Unexpectedly, results from hair follicle analyses and fibroblast selection studies in 8-azaguanine and 6-thioguanine medium showed a non-carrier phenotype in three of the female heterozygotes, whereas X-inactivation mosaicism was demonstrated in one heterozygote. A possible explanation for the apparent non-random X-inactivation in this family is the co-existence of the hprt mutation with an undefined X-linked lethal mutation. This observation is of practical relevance for carrier detection in other Lesch-Nyhan families.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.