Oriented thick and thin filaments in amoeba proteus

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.     

Hydrolysis of neutral glycerides by lipases of rat brain microsomes.

The hydrolysis of monoacylglycerol and diacylglycerol by rat brain microsomes was followed by measuring the release of glycerol and monooleylglycerol from dispersions of water insoluble glyceryl esters of oleic acid. The microsomes showed three lipolytic activities. One activity, optimal at pH 4.8, catalyzed the hydrolysis of diacylglycerol but not monoacylglycerol. Two other lipolytic activities, optimal at pH 8.0-8.6, catalyzed the hydrolysis of both diacylglycerol and monoacylglycerol. The pH 8.0-8.6 activities were sensitive to heat and SH-reagents. Detergents were inhibitory in all cases. Extraction of the microsomes with KCl, KSCN, urea or Triton X-100 did not change the ratio of diacylglycerol hydrolysis at pH 4.8 and 8.0. The results of subcellular fractionation studies showed that there was no significant enrichment of the acid lipase in any fraction.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

The hydrolysis of triacylglycerol and diacylglycerol by a rat brain microsomal lipase with an acidic pH optimum.

Lipase activity towards triacylglycerol and diacylglycerol was measured at pH 4.8 using a microsomal preparation from rat brain as the enzyme source. The optimal pH for the hydrolysis of triacylglycerol was 4.8, with only minor lipolytic activity in the alkaline pH range. Diacylglycerol was the major product of triacylglycerol hydrolysis, with only little monoacylglycerol being formed. When diacylglycerol was the starting substrate it was hydrolyzed at a rate 10-fold greater than triacylglycerol, and the product was monoacylglycerol. The enzyme showed positional specificity for the fatty acid moieties located at the primary positions of sn-glycerol. 1,3-Diacylglycerol was hydrolyzed at greater than twice the rate of the corresponding 1,2(2,3)-isomer.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.     

Phylogenetic analyses of the rbcL sequences from haptophytes and heterokont algae suggest their chloroplasts are unrelated

Using the large subunit of RuBisCo (rbcL) sequences from cyanobacteria, proteobacteria, and diverse groups of algae and green plants, we evaluated the plastid relationship between haptophytes and heterokont algae. The rbcL sequences were determined from three taxa of heterokont algae (Bumilleriopsis filiformis, Pelagomonas calceolata, and Pseudopedinella elastica) and added to 25 published sequences to obtain a data set comprising 1,434 unambiguously aligned sites (approximately 98% of the total rbcL gene). Higher levels of mutational saturation in third codon positions were observed by plotting the pairwise substitutions with and without corrections for multiple substitutions at the same site for first and second codon positions only and for third positions only. In accordance with this finding phylogeny reconstructions were completed by omitting third codon positions, thus using 956 bp in weighted-parsimony and maximum-likelihood analyses. The midpoint-rooted phylogenies showed two major clusters, one containing cyanobacteria, glaucocystophytes, a phototrophic euglenoid, chlorophytes, and embryophytes (the green lineage), the other containing proteobacteria, haptophytes, red algae, a cryptophyte, and heterokont algae (the non-green lineage). In the nongreen lineage, the haptophytes formed a sister group to the clade containing heterokont algae, red algae, and the cryptophyte Guillardia theta. This branching pattern was well supported in terms of bootstrap values in weighted- parsimony and maximum-likelihood analyses (100% and 92%, respectively). However, the phylogenetic relationship among red algae, heterokonts, and a cryptophyte taxon was not especially well resolved. A four- cluster analysis was performed to further explore the statistical significance of the relationship between proteobacteria, red algae (including and excluding Guillardia theta), haptophytes, and heterokont algae. This test strongly favored the hypothesis that the heterokonts and red algae are more closely related to each other than either is to proteobacteria or haptophytes. Hence, this molecular study based on a plastid-encoded gene provides additional evidence for a distant relationship between haptophytes and the heterokont algae. It suggests an evolutionary scenario in which the ancestor of the haptophyte lineage engulfed a phototrophic eukaryote and, more recently, the heterokont lineage became phototrophic by engulfing a red alga.      

Structure of replicating hepatitis C virus (HCV) quasispecies in the liver may not be reflected by analysis of circulating HCV virions.

We have analyzed the population of hepatitis C virus (HCV) sequences in paired liver and serum samples from four patients with chronic hepatitis C. Sequences from three different biopsy specimens from a liver explant from one patient were compared with each other and with the circulating sequences. Our results demonstrate that the circulating quasispecies does not necessarily reflect the viral population replicating in the liver and that this is not due to a macroscopic anatomic compartmentalization of HCV replication. This finding has important implications for the pathogenesis and natural history of chronic HCV infection.     

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.     

Variation in the harvest index of tropical maize: evaluation of recent evidence from Mexico and Malawi

In temperate zones, the potential grain yield of wheat has increased during the twentieth century owing to progressive increases in the harvest index of new varieties, which are principally associated with reduction in plant stature. Crop biomass has not increased substantially. In contrast, the potential grain yield of maize in the USA has increased owing to progressive increases in biomass, principally associated with selection for grain yield at higher population density. Harvest index was already around 0.5 for recommended varieties in 1930, and has not increased significantly since. However, for both crops, the harvest index of a given variety has proved to be a highly‐heritable character, except under severe stress. Less is known about the physiology of tropical maize. This paper reviews evidence from Mexico and Malawi that tropical maize can respond to selection for reduced stature following the same pattern as temperate wheat, but, under other circumstances, the magnitude of harvest index is not highly heritable, varying inconsistently with season, management and environment. It is proposed that these differences arise out of the unique vulnerability of the grain‐setting process around flowering. The plasticity of harvest index under long, favourable conditions, however, remains to be explained, although it is probably also related to the events around grain setting. Nevertheless, to the subsistence farmer, higher harvest index may not be a high priority in crop improvement, because of the need for large quantities of high‐quality stover.     

Morphological Constraints of Shoot Demography of a Clonal Plant: Extra- and Intravaginal Tillers of Festuca rubra

Abstract Festuca rubra forms tillers in two different ways: extravaginally and intravaginally. Demography of these two tiller types was observed in seventeen selected tussocks of Festuca rubra s.s. over four growing seasons. Extravaginal tillers were bigger at birth and on the average produced twice as many daughter tillers per tiller. In general, the natality and mortality of extravaginal tillers were less regular than that of intravaginal tillers. Overall tillering rate per tiller was correlated with the density of the surrounding vegetation; mortality, natality and tiller life span were not. High density of the surrounding vegetation did not result in increased formation of extravaginal tillers. The proportion of the extravaginal tillers was not correlated with the density of the F. rubra tussocks. There is no evidence for foraging by extravaginal tillers, but they do act as founders of small clusters of tillers.