Action of a microbial glycerophospholipid:cholesterol acyltransferase on plasma from normal and LCAT-deficient subjects

The action of a bacterial acyltransferase similar in overall reaction mechanism to the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been studied using normal plasma and lipoproteins and plasma from LCAT-deficient patients. The microbial enzyme (GCAT) catalyzed acyl transfer using phosphatidylcholine and cholesterol in all of the lipoprotein fractions, presumably because it has no apolipoprotein cofactor. In addition, the enzyme was capable of hydrolyzing cholesteryl ester in lipoproteins but not in small unilamellar vesicles nor in micellar dispersions containing low amounts of Triton X-100. This suggests that cholesteryl ester is exposed on the surface of lipoprotein particles or that it may be transferred there quickly from the interior. Although considerable interconversion of radiolabeled cholesterol and cholesteryl ester could be demonstrated upon treatment of normal plasma or lipoproteins with the enzyme, there was little change in the actual amount of either steroid. This indicates that the rate of cholesteryl ester formation is very similar to the rate of hydrolysis. The relative proportions of cholesterol and cholesteryl ester in normal plasma are therefore near the equilibrium ratio for the reaction carried out by GCAT, or the ratio is controlled by the properties of the lipoproteins themselves. During reaction with the microbial acyltransferase, the ratio of cholesterol to cholesteryl ester in plasma from LCAT-deficient patients was reduced substantially, suggesting that the enzyme may have some practical applications.     

Monoclonal antibodies to plant plasma-membrane antigens

Murine monoclonal antibodies to membrane antigens were generated by immunization with a crude cellular membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. From a panel of thirteen monoclonal antibodies, seven were found to be directed against antigens present on the plasma-membrane by immunofluorescence visualization of antibody binding to the surface of isolated protoplasts. The corresponding set of plasma-membrane antigen(s) were present in root, shoot and leaf tissue and some but not all of these antigens were of wide species distribution, being found in Nicotiana tabacum L., N. plumbaginifolia L., Glycine max L., Phaseolus vulgaris L. and Triticum aestivum L. Topologically specific labeling of intact protoplasts with a monoclonal antibody reactive with an epitope present on the plasma-membrane specifically labeled a membrane fraction which equilibrated at a density of 1.14 kg/l following centrifugation in a sucrose gradient. In addition to use as biochemical markers for fractionation and molecular characterization of plasma-membranes, these monoclonal antibodies provide the basis for new selection tools in plant cell and gene manipulations.     

Chlorophyll-protein and polypeptide composition of Mn-deficient sugar beet thylakoids

The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.     

Variation in photosynthetic electron transport capacity in vivo and its effects on the light modulation of ribulose bisphosphate carboxylase

Photosynthetic electron transport capacity was varied in vivo in sugar beets using iron deficiency, and its effects on the light modulation of ribulose bisphosphate carboxylase (RuBPCase) studied. Three treatment groups corresponding to decreasing amounts of thylakoids per leaf area were examined: iron sufficient (control), moderately iron-stressed, and severely iron-stressed. Reduction in electron transport capacity in vivo was correlated with a substantial decrease in the level of RuBPCase activation, even at saturating irradiances. These results indicate a direct relationship between RuBPCase activation and photosynthetic electron transport. In addition, our data suggest that the activation of RuBPCase could not solely account for the increases in the photosynthetic rate at high irradiances; RuBPCase reached maximal activation at irradiances well below light saturation for net photosynthesis.Abbreviations Chl chlorophyll - FeCN ferricyanide - FBPase fructose 1,6-bisphosphatase - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose 1,5-bisphosphate carboxylase - SBPase sedoheptulose 1,7-bisphosphatase     

Tripartite sequences within and 3'' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human alpha-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human alpha-globin gene. This provides evidence for the role of poly(A) in the stability of mRNA.     

Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle.

The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid. In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo. We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it. This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski, Cell 40:795-803, 1985; J. F. Senecoff, R. C. Bruckner, and M. M. Cox, Proc. Natl. Acad. Sci. USA 82:7270-7274, 1985). Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines. However, mutations outside the core are not similarly suppressed. We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate. Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency. The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.     

Respiratory function of children in homes insulated with urea formaldehyde foam insulation.

A study was carried out to assess the respiratory function of children living in homes insulated with urea formaldehyde foam insulation (UFFI). A large data base on the effect of environmental variables on the respiratory function of 3500 children in the Hamilton, Ont., area had been collected from 1978 to 1980. From this data base 29 children who lived in UFFI-insulated homes were identified, and each was matched with 2 controls according to nine variables that had been shown to be strongly predictive of respiratory function. Reported respiratory symptoms and results of pulmonary function testing in the year immediately following installation of UFFI were examined. No significant differences in any variable were found between the subjects and controls. A power calculation indicated that the study had adequate power to detect clinically important changes. The authors conclude that there was no evidence of respiratory problems resulting from UFFI in the sample studied.     

Feasibility of automating the micronucleus assay

The results of a feasibility study on the automation of the micronucleus assay in whole blood cultures of human lymphocytes are reported. The assay requires determination of the number of lymphocytes with micronuclei among the proliferating population. Using an in-house-assembled image analysis system, a prototype software package was developed that addressed two problems: micronuclei identification and discrimination of nonproliferating cells from proliferating lymphocytes (the only ones that can give rise to micronuclei). The results of manual verification of automated micronucleus scoring showed that 70% of all digitized micronuclei were extracted from the images and 90% of them were correctly classified and paired with a parent nucleus by an "affinity function". The discrimination between proliferating and nonproliferating cells was carried out by linear discriminant analysis of simple nuclear features extracted from Feulgen-stained cells. Among the Feulgen-stained nuclei that were identified by autoradiography as proliferating or not, 85% were correctly classified by a six-feature discriminant function.     

The effects of aluminum loading on the renal response to parathyroid hormone in the vitamin D-replete rat

Aluminum (Al) may cause vitamin D-resistant osteomalacia and depress the serum levels of immunoreactive parathyroid hormone (iPTH) in patients treated with maintenance dialysis and those on total parental nutrition (TPN). Both conditions have been associated with low serum levels of 1,25(OH)2-vitamin D (1,25(OH)2D). Al may inhibit PTH secretion in vitro; however, induction of hypocalcemia can enhance endogenous PTH secretion in Al-loaded dogs and TPN patients. Despite hypocalcemia and/or increased endogenous iPTH levels, Al-loaded TPN patients fail to show the expected rise in serum 1,25(OH)2D levels. Such observations suggest that Al may impair the renal response to PTH. We studied vitamin D-replete rats given Al or saline vehicle IP for 5 days. Al and control rats then received a saline infusion with an IV bolus of PTH 1-34. Urinary cyclic AMP and P excretion rose in Al and control rats by 1 hr post-PTH, without differences between the groups. Serum P and ionized Ca levels were not different between Al and control rats. In other Al and control rats, serum 1,25(OH)2D levels were measured after saline without PTH. Serum 1,25(OH)2D levels were higher in controls given PTH than in those without, but 1,25(OH)2D levels were not different between Al rats given PTH and those with none. Thus, aluminum does not affect cyclic AMP or P excretion but may impair 25(OH)D-1 alpha-hydroxylase activity in response to PTH.