Phospholipid methyltransferase activity in pancreatic islets: activation by calcium

Pancreatic islet homogenates contain a Mg2+-requiring phospholipid methyltransferase activity, the activity of which was doubled by calcium (K0.5 less than 5 microM). Other divalent metal ions stimulated the activity from 11 to 35%, but zinc and strontium were inhibitory. Cyclic AMP had no effect on the enzyme activity and cyclic GMP inhibited it slightly. Calcium increased the Vmax of the enzyme without affecting its Km with respect to S-adenosylmethionine (6 microM). Chlorpromazine, trifluoperazine, and dibucaine inhibited the calcium-stimulatable activity without affecting the activity in the absence of calcium. Phosphatidylserine stimulated, and arachidonic acid and palmitic acid inhibited, the basal enzyme activity. The methylated products were found to be primarily mono- and dimethylphosphatidylethanolamine (30%) and phosphatidylcholine (43%) and an, as yet unidentified, nonpolar lipid fraction (27%), as judged by thin-layer chromatography. In the presence of calcium, incorporation of methyl groups into phosphatidylcholine, mono- and dimethylphosphatidylethanolamine, and nonpolar lipids was increased by 131, 60, and 46%, respectively. Based on the localization of the enzyme activity in the insulin secretory granule fraction, it is proposed that phospholipid methylation plays a role in coupling the stimulus to the initial events in insulin secretion, leading to the exocytosis of insulin.     

An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.     

Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.     

Modulation of rat granulocyte traffic by a surface active agent in vitro and bleomycin injury

Pluronic F68 (F68) is a nonionic surfactant which has been reported to inhibit the in vitro adherence and migration of polymorphonuclear leukocytes (PMN) obtained from some species. We demonstrated similar effects on PMN obtained from rats, with diminished adherence to nylon wool and diminished chemotaxis toward zymosan-activated serum. We then examined the in vivo effects of 12-hr F68 infusion on the injury induced by intratracheal bleomycin instillation (ITB) in rats. When sacrificed 24 hr following injury, rats demonstrated neutrophilia, neutrophil-prominent lung lavage cellularity, and increased lung weights. F68 decreased lavage leukocyte counts and lung weight gain in ITB-injured animals. Lung weights of ITB-injured animals correlated (r = 0.81, P less than 0.001) with logarithmic values of lavage PMN. F68 also enhanced neutrophilia and decreased spleen weight gain in injured animals. The acute effects of F68 on circulating leukocyte counts, osmolality, and total complement were also examined. The data demonstrate that F68 can affect PMN traffic both in vitro and in vivo. The data also confirm the prominence of PMN in lavage fluid early in ITB injury, and suggest that an influx of relatively few PMN is associated with lung weight gain in this model.     

Cell kinetics of vocal fold epithelium in rats.

In order to investigate the kinetics of vocal fold epithelium a bromodeoxyuridine-anti bromodeoxyuridine method has been applied in vivo at both light and electron microscopy level. This method is able to define the length of both epithelium turnover and cell-cycle in basal elements, as well as the existence of a higher proliferation rate during night time in comparison with day time. Moreover distinct labeling patterns observed in incorporating cells allow us to define the precise localization in S-phase of cycling elements.     

A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.